The effect of carbon tetrachloride on the total plasma protein concentration of rainbow trout, Salmo gairdneri

Comp. Biochem. Physiol., Vol.64C,pp. 37 to 42

0306-4493/79/0801-0037g02.00/0

© PergamonPressLtd 1979.Printedin GreatBritain

THE EFFECT OF CARBON TETRACHLORIDE ON THE TOTAL PLASMA PROTEIN CONCENTRATION OF RAINBOW TROUT, SALMO G A I R D N E R I KEITH F. PFEIFER and LAVERN J. WEBER

Department of Fisheries and Wildlife, Oregon State University, Corvallis, Oregon 97331, U.S.A. (Received 27 February 1979) Abstract--1. The effect of a single i.p. injection of CCI, on total plasma protein and albumin concen-

trations was measured in the rainbow trout. 2. CC14 treatment caused a significant reduction in total plasma protein concentration at 12, 24 and 36 hr post-treatment. 3. At 24 hr post-treatment plasma albumin concentration was significantly reduced only in fish given the highest dose of CCI, (2.0 ml/kg). 4. Several factors, including intestinal inflammation and hemorrhages, could have contributed to the decreased plasma protein concentration in the trout.

INTRODUCTION

Smith, 1971; Hunn, 1964; Mulcahy, 1969). Zeitoun (1977) determined the acute effect of chlorine on several blood parameters of rainbow trout and found a significant increase in plasma protein concentration. Except for the study by Zeitoun, there is little information currently available regarding the effects of chemicals on fish plasma proteins. The objective of this study was to determine the effect of CC1, treatment on the plasma protein concentration of rainbow trout, Salmo gairdneri.

Carbon tetrachloride (CC14) has become a model hepatotoxic agent in mammalian experimental toxicology because of its ability to cause reproducible liver damage (Raisfeld, 1974). Two characteristic and apparently separable responses of CC14 intoxication are hepatic lipid accumulation and necrosis. Previous studies with mammals established that CC14 can depress hepatic protein synthesis (Recknagel & Ghoshal, 1966; Smuckler et al., 1962; Smuckler & Benditt, 1965); however, Recknagel (1967) concluded that CCla-induced depression of protein synthesis was not the primary event leading to hepatic lipid accumulation or hepatic necrosis. Smuckler et al. (1961) showed that CC14 reduced the incorporation of amino acids into plasma proteins in laboratory rats. Earlier reports indicated that CC14 can decrease the total plasma protein concentration in laboratory mammals (Berryman & Bollman, 1943; Erickson et al., 1938). Previous studies established that rainbow trout are susceptible to some of the hepatotoxic effects of CC1, reported for mammals. Pfeifer et al. (1977), Racicot et al. (1975) and Statham et al. (1978) recorded increased plasma activities for enzymes, presumably of liver origin, after treatment with CC14. Gingerich et al. (1978a) demonstrated an increase in plasma sulfobromophthalein retention following CC14 treatment. Gingerich et al. (1978a), Racicot et al. (1975) and Statham et al. (1978) reported varying degrees of hepatic necrosis in CC1,-treated rainbow trout; however, in contrast to mammalian findings, Statham et al. (1978) concluded that there was no increase in hepatic triglyceride concentrations. The plasma protein concentration of teleosts can be affected by: season of the year (Denton & Yousef, 1975); stage of life cycle (Robertson et al., 1961a, b; Triplett & Calaprice, 1974); diet (Koroleva, 1964); environmental temperature, photoperiod or oxygen concentration (Bouck & Ball, 1965; Guderly & Ulrickson, 1973; Meisner & Hickman, 1962; Poston, 1965; Umminger, 1970); and disease states (Cardwell &

MATERIALS AND METHODS

Rainbow trout (100-250 g) were purchased from Roaring River Hatchery (Oregon Department of Fish and Wildlife), Scio, Oregon and held in aquaria (200 gal) supplied with continuously flowing well water (15.0°C + 1.0°). A 12L:12D photoperiod was used throughout all experiments. Animals were fed a commercial fish diet, Donaldson-Ore Aqua, every day, but food was withheld for 24 hr prior to an experiment. All fish were in good health and allowed a one-week acclimation period before they were used. The study was conducted during the months of September through November. Twenty-four hour "fasted" fish were weighed, fin marked and placed in 50gal aquaria supplied with continuously flowing well water (15.0°C + 1.0°). After 24 hr the fish were given an intraperitoneal (i.p.) injection of undiluted CC14. Control fish received an equivalent volume of Cortland saline (Wolfe, 1963). In the initial experiment the fish were treated with CC14 (2.0 ml/kg). Blood samples were obtained from the caudal vein at 3, 6, 12, 18, 24 and 36hr post-treatment using a heparinized syringe and needle. The plasma was separated from blood cells by centrifugation and kept at 0-4°C until analyzed for total protein concentration and osmolality. The subsequent experiment measured total plasma protein and albumin concentrations at 24hr post-treatment with CC14 administered at 0.25, 0.50, 1.0 or 2.0ml/kg. Nontreated control fish were sampled directly from the holding aquaria and not subjected to the experimental procedure described above. Final body weight for control or CCI,treated fish was determined prior to obtaining the blood sample. 37

KEITH F. PFEIFERand LAVERNJ. WEBER

38

Plasma protein concentration was measured by the biuret method (Gornall et al., 1949) and corrected for plasma hemoglobin concentration by the method outlined in Sigma Bulletin No. 540. Plasma hemoglobin concentration was determined by the cyanmethemoglobin method of Richterich (1969). Plasma osmolality was measured with a Wescor vapor pressure osmometer. Plasma albumin was determined by the method in Sigma Bulletin No. 630 which is based on the affinity of albumin for bromcresol green. Experimental data were converted to a quantal form and analyzed by the Fisher Exact Probability Test (Siegel, 1956). Values greater than two SD (P < 0.05) from the mean non-treated control value were chosen to indicate a positive response in the experimental fish. At each sampling time three CC14-treated and one saline control fish were killed by a blow to the head, and a portion of the trunk kidney was immediately removed from each fish. Kidney sections were fixed in Bouin's solution, stained with hematoxylin and eosin and evaluated for morphological changes. RESULTS

The plasma protein concentration of CC14-treated rainbow trout was significantly different (P < 0.05) from control fish as early as 12 hr post-treatment and at 36hr was 55~o less than the mean control value (Fig. 1). Carbon tetrachloride given at 0.25 or 0.5 ml/kg did not significantly decrease (P ~< 0.05) the total plasma protein or albumin concentrations at 24 hr post-treatment (Fig. 2); however, trout treated with CC14 at 1.0 ml/kg had a significant reduction (P ~< 0.05) in total plasma protein but not in albumin concentration. Carbon tetrachloride given at 2.0 ml/kg produced a highly significant decrease (P < 0.01) in both the total plasma protein and albumin concentrations. Total plasma protein and albumin concentrations for non-treated control fish were 27.9mg/ml and 13.7 mg/ml, respectively (Fig. 2). These values were similar to those obtained for the saline-treated control fish. Fish treated in CC14 (2.0ml/kg) had an absolute (actual) and relative (g/100g B.W.) weight gain starting at 12 hr post-treatment and continuing

for the remaining sampling times, i.e. 18-48 hr (Fig. 3). The maximum relative weight gain was 6.4 g/100 B.W. and occurred at 36hr post-treatment. Control fish, on the other hand, decreased in absolute and relative body weight at each sampling time. Plasma osmolality for CCl4-treated fish was less than Cortland controls at all sampling times, except 12hr (Fig. 4). The lowest osmolality for treated fish was 268 mOs/kg and occurred at 36 hr post-treatment. This osmolality was significantly different (P < 0.01) from the 36 hr mean control value. Histological examination of kidney sections from the CC14-treated fish did not provide evidence of consistent morphological pathology although some cellular damage was observed. Hematopoietic cell necrosis, characterized by pyknosis or karyorrhexis, occurred in CCI4-treated fish at 3 and 6 hr post-treatment. Subtle changes, such as alterations in the brush border of the first and second proximal tubule segments were observed in one fish at 24 hr post-treatment. Another fish at 36 hr had severe microdroplet degeneration of the first proximal tubule segment. There was no temporal pattern to the resultant histopathology produced by the CC14. No damage to glomeruli, distal tubule segments or collecting ducts was noted in any CC14-treated fish. DISCUSSION

Plasma protein synthesis in mammals occurs primarily in the liver. Investigations by Kenyon (1967) and Dean & Berlin (1969) suggested that the liver of teleosts has a similar capability; however, the functional significance of teleostean plasma proteins is unclear. (Houston, 1973) suggested that plasma proteins of these fish are important in cardiovascular adjustments to temperature changes and ~i~-,i,mmune response mechanisms. In order to maintain ~in effective plasma osmotic pressure, higher vertebrates, e.g. humans, have a plasma protein concentration approximately twice that found for trout in our study. The mean arterial systolic blood pressure in humans is 16kPa. Rainbow trout, on the other hand, have a systolic pressure in the dorsal aorta of approx

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Fig. 1. Plasma protein concentration for rainbow trout treated with Cortland saline or CCI4 12.0ml/kg, i.p.). Values are the mean + SE for the number of fish in parentheses. Zero time mean value is for non-treated control fish. Asterisks denote values that are significantly different (*P < 0.05, **P < 0,011 from Cortland-treated control fish.

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Fig. 2. Total protein and albumin concentrations in plasma from rainbow trout 24hr post-treatment with Cortland saline or CC14 (0.25 ml, 0.5 ml, 1.0 ml or 2.0 ml/kg, i.p.). Values are the mean _+SE for the number of fish in parentheses. Cortland control concentrations are the mean of pooled values at the four doses. Non-treated control plasma concentrations were determined for fish not subjected to the injection procedure. Asterisks denote values that are significantlydifferent (*P < 0.05, **P < 0.01) from mean concentrations of Cortland-treated fish.

4.6 kPa (Randall et al., 1965). The lower blood pressure in the rainbow trout apparently indicates a reduced plasma protein requirement to maintain blood flow and organ perfusion. The trout treated with CC14 (2.0 ml/kg) had a 50% decrease in total plasma protein concentration at 24 hr but were alive and swimming. A comparable plasma protein reduction in humans would have been followed by vascular collapse, renal failure and death. The increase in body weight and decrease in plasma osmolality in CCl4-treated fish suggested that water was retained and that all or part of the plasma protein reduction was due to an increase in plasma volume. Blood or plasma volume changes were not established; however, an in vitro dilution of trout plasma having a known protein concentration and osmolality indicated that approx 20% of the total pro-

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Fig. 3. Relative body weight changes for rainbow trout treated with Cortland saline or CC14 (2.0ml/kg, i.p.). Values are the mean + SE for the number of fish in parentheses. Asterisks denote values that are significantly different (*P < 0.05, **P < 0.01) from Cortland-treated control fish.

tein decrease at 24 hr could be attributed to plasma dilution. Fish given CC14 (1.0 or 2.0 ml/kg) had a decrease in plasma albumin concentration at 24 hr, but the increase in the albumin/total plasma protein ratio indicated that a part of the reduction was presumably from a loss of some other plasma protein fraction, e.g. globulins. Erickson et al. (1938) found that the plasma proteins in CC14-poisoned dogs were reduced mainly by a decrease in the albumin fraction. Additionally, Berryman & Bollman (1943) reported a reduction of total plasma proteins, chiefly albumin, and a relative and absolute increase in the globulin fraction in CCl4-treated laboratory rats. However, the implication that a specific protein fraction accounted for the plasma protein reduction in our fish is difficult because of the complex homeostatic mechanisms (in higher vertebrates) which control the level of plasma proteins (Zilva & Pannall, 1972). Histopathological examination of kidney tissue failed to establish consistent morphological damage in the CC14-treated fish. No glomerular damage was observed, and only one kidney had proximal tubular necrosis. Hematopoietic cell necrosis and early pathological evidence, such as alteration in the brush border of the proximal tubules, may have been indicative of other subtle changes in nephron organelle morphology that were not apparent with the light microscope. Non-treated control fish had a mean plasma protein concentration lower than reported values in previous studies with rainbow trout, and plasma albumin concentration represented a greater percentage of the total plasma proteins in our trout. Sauer & Haider (1977) reported a plasma protein concentration of approx 34mg/ml for rainbow trout at 15°C. Denton & Yousef (1975) recorded a plasma protein concentration of 46mg/ml for rainbow trout during November (water temperature: 11-13°C). Zeitoun (1977), on the other hand, reported a mean total plasma protein concentration of 64 mg/ml in rainbow

40

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Fig. 4. Plasma osmolality for rainbow trout treated with Cortland saline or CC14 (2.0 ml/kg, i.p.). Values are the mean + SE for the number of fish in parentheses. Zero time mean value is for non-treated control fish. Asterisks denote values that are significantly different (*P < 0.05, **P < 0.01) from Cortland-treated control fish. trout. Meisner & Hickman (1962) determined that the plasma albumin concentration of rainbow trout at 16°C was 44~ of the total plasma proteins, while Guderley & Ulrickson (1973) found plasma albumin was 29~ of total plasma protein. The apparent discrepancy in plasma protein and albumin concentrations in rainbow trout could be attributed to season of year, water temperature, strain of fish, diet or analytical methods. The reduced plasma protein concentration in the CCl4-treated fish could be attributable to several pathological processes, including the following: in vivo plasma dissolution, renal damage and elimination in the urine, decreased liver protein synthesis, alteration in hepatic blood flow and/or hemorrhages into the peritoneal cavity and intestine. It is unlikely that the decreased plasma protein concentration could be attributed to an in vivo plasma protein degradation by CC14. Concurrent with the reduction in plasma protein concentration in the CCl4-treated trout was an increase in plasma enzyme activity, e.g. alanine aminotransferase (GPT). The G P T activity was maximum at 36 hr post-treatment (Pfeifer, 1979). Curtis et al. (1972) reported that CC14 had no effect on mammalian plasma GPT activity in vitro; therefore, CC14 would appear incapable of a selective in vivo plasma protein, i.e. albumin, dissolution to account for the reduction in plasma protein concentration in our trout. Carbon tetrachloride has been shown to be nephrotoxic to mammals (Moon, 1950; Stricker et al., 1968). The renal damage was confined mainly to tubule portions of the nephron, but albuminuria has been confirmed in some cases of CC14 toxicity (Von Oettingen, 1955), indicating possible glomerular damage. Histopathological examination of kidney tissue from our CCl4-treated trout showed no glomerular damage, and only one fish had proximal tubule necrosis. Preliminary work using a dipstick test (Albustix ®) for proteinuria indicated that trace amounts of protein (<80 mg/100 ml) appeared intermittently in the urine during the 24hr post-treatment with CC14. These results and the lack of demonstrable histopathologic

alteration suggested that significant amounts of plasma proteins were not lost through renal elimination. The majority of plasma protein synthesis in rainbow trout presumably occurs in the liver (Dean & Berlin, 1969; Kenyon, 1967). Smuckler & Benditt (1965) concluded the decrease in liver and plasma protein synthesis in laboratory rats given CCI4(PO) resulted from a change in ribosome structure. Previous studies by these workers showed that labeled amino acid incorporation into fibrinogen and albumin was depressed as early as 3 and 4 hr, respectively, post-treatment with CC14, but plasma protein concentration and relative plasma protein fractions were not altered during this period (Smuckler et al., 1961, 1962). The plasma half-life for proteins, e.g. albumin, in rainbow trout is not presently known; however, it would appear unlikely that the significant reduction in total plasma protein experienced by our trout at 24 hr and 36 hr could be attributed entirely to a decrease in plasma protein synthesis. The reduction in plasma proteins may have resulted from an impairment in hepatic blood flow. A decrease in amino acid availability and/or in hepatic protein secretory capability would reduce the total plasma protein concentration. Gingerich et al. (1978b) suggested that a decrease in hepatic blood flow may have been responsible for impaired plasma clearance and hepatic accumulation of sulfobromophthalein in CC14-treated rainbow trout; however, information about hepatic blood flow and the circulatory changed produced by C C I 4 in fishes is not presently available. Rainbow trout given CC14 by i.p. injection showed severe inflammation of the peritoneal cavity and general soft organ damage. Hemorrhagic fluid was characteristically present in the peritoneal cavity, and the spleen and intestines were consistently damaged. Additionally, the fish experienced a progressive inflammation of the urogenital opening and expelled small pieces of hemorrhagic tissue. Previous work with laboratory mammals indicated that an i.p. injection of CC14 can cause an effusion of hemorrhagic fluid into the peritoneal cavity (Von Oettingen, 1955).

Effect of CC14 on trout plasma protein Bollman et al. (1940) reported that hemorrhages were present in the gastro-intestinal tract of rats given CC14 by inhalation. They found that the blood of the rats had an increased prothrombin time and concluded that the decreased clotting ability and hemorrhages were contributing factors in the death of the animals. Additionally, CC14 can directly damage capillaries leading to diapedesis (Von Oettingen, 1955). The cited examples from mammalian toxicological studies, the observable gross pathology and the significant early reduction in plasma protein concentration found in CC14-treated rainbow trout indicate that a probable loss of plasma protein occurred from hemorrhages into the peritoneal cavity or through the gastro-intestinal tract. Thus, several factors could have contributed to the decrease in plasma protein concentration in the CCl4treated rainbow trout. Plasma protein loss may have occurred in the urine due to renal damage. The hepatotoxic effects of CC14 may have resulted in decreased plasma protein synthesis or impaired hepatic blood flow. A decrease in prothrombin synthesis and direct capillary damage would have permitted plasma protein loss from hemorrhages into the peritoneal cavity or through the gastro-intestinal tract. Further studies are needed to establish the relationship and relative importance of these processes in CC14 intoxicated animals. Acknowledgements--The authors thank Dr Robert Larson and Mr Wayne Seim for reviewing the manuscript and Dr Jerry Hendricks for his help with the histological studies. Support for this work was provided in part by a training grant GMO 7148 from the National Institutes of Health and from a research grant R 803090 from the United States Environmental Protection Agency. Oregon Agricultural Experiment Station Technical Paper No. 5094. REFERENCES

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KEITH F. PFEIFER and LAVERN J. WEBER

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STATHAM C. N., CROFT W. A. & LECH J. J. (1978) Uptake, distribution and effects of carbon tetrachloride in rainbow trout, Salmo gairdneri. Toxicol. appl. Pharmac. 45, 131-140. STR1CKER G. E., SMUCKLERE. A., KOHNEN P. W. & NAGLE R. B. (1968) Structural and functional changes in rat kidney during carbon tetrachloride intoxication. Am. J. Path. 53, 769-789. TRIPLETT E. & CALAPRICE J. R. (1974) Changes in plasma constituents during spawning migration of Pacific salmon. J. Fish Res. Bd. Can. 31, l 1-14. UMMINGER B. L. (1970) Effects of temperature on serum protein components in the killifish, Fundulus heteroclitus. J. Fish. Res. Bd Canada 27, 404409. VON OETTINGEN W. F. (1955) The Halogenated Hydrocarbons, Toxicity and Potential Dangers, pp. 75-112. Public Health Service Publ. No. 414. U.S. Govnt. Printing Office, Washington, D.C. WOLFE K. (1963) Physiological salines for freshwater teleosts. Prog. Fish Cult. 25, 135-140. ZEITOUN I. H. (1977) The effect of chlorine toxicity on certain blood parameters of adult rainbow trout, Salmo gairdneri. Envir. Biol. Fish. l, 189 195. ZILVA J. F. • PANNALL P. R. (1972) Clinical Chemistry in Diagnosis and Treatment, pp. 227-247. Year Book Medical Publishers, Chicago, Ill.