Mary F. Kanz

Ninety Day Toxicity Study of Chloroacetic Acids in Rats

Chloroacetic acids are produced in drinking water as a result of disinfection processes. Chloroacetic acids are also metabolites of widely used and toxic halogenated hydrocarbons. Thus, chronic human exposure to these chemicals is likely to occur. The objective of the present study was to examine the toxic effects of monochloroacetic acid (MCA), dichloroacetic acid (DCA), and trichloroacetic acid (TCA) in a 90-day subchronic study in rats via oral exposure by drinking water. Chloroacetic acid solutions were prepared at concentrations which provided an approximate intake of 1/4 the LD50 dose per day: MCA, 1.9 mM; DCA, 80.5 mM; TCA, 45.8 mM. Control rats received distilled water only. After 90 days, major organs were removed, fixed, paraffin embedded, and stained. Light microscopic examination of the major organs revealed variable degrees of alterations in the lung and liver of all three treated groups. In the liver, morphological changes were predominantly localized to the portal triads, which were mildly to moderately enlarged with random bile duct proliferation, extension of portal veins, fibrosis, edema, and occasional foci of inflammation. In the lungs, minimal alterations were observed as foci of perivascular inflammation on small pulmonary veins. Morphological changes in the testes and brain were seen only in the DCA treated group. Testes were atrophic with few spermatocytes and no mature spermatozoa. Focal vacuolation and gliosis were present in the forebrain and brainstem. The results of these studies indicate that, relative to their respective LD50 values, DCA given at 80.5 mM is more toxic than TCA given at 45.8 mM and MCA at 1.9 mM is least toxic.

A stable colorimetric assay to measure toxin elevation of inorganic phosphate in bile

A modified assay for the measurement of nanomole amounts of Pi in 50 microliters of bile is described. The assay is based on the formation of a complex between malachite green dye and phosphomolybdate under acidic conditions. Only three simple steps are required to produce a colored complex which remains stable for at least 3 h; these steps are precipitation with acid, addition of color reagent containing dye and surfactant, and incubation at 37 degrees C. Analysis of bile samples collected from a rat given the toxin 1,1-dichloroethylene demonstrated that the assay is well suited to routine assays of biliary Pi as an endogenous indicator of aberrant hepatobiliary function.

Biliary function studies during multiple time periods in freely moving rats a useful system and set of marker solutes

Biliary output of endogenous and exogenous compounds is altered by anesthesia, depletion of bile salts, and hydrostatic pressure. The described system for bile function studies minimizes these confounding factors by substantially modifying existing methods. Experiments were conducted in freely moving rats which eliminates effects of anesthesia or restraint-induced stress. Depletion of bile salts was prevented by intraduodenal infusion of taurocholate which maintains bile volume. Bile was collected in containers taped to the rats back which minimizes hydrostatic forces induced by lengthy or elevated biliary cannulas. Animals were prepared for hepatobiliary function studies 1 week before experiments by placement and exteriorization of a jugular cannula and a bile duct to duodenal fistula. Experiments involved monitoring biliary outputs of marker solutes for various pathways of bile formation during three sequential time periods of 120 min, that is, a basal period in the morning and two experimental periods in the afternoon. We found similar patterns of biliary output in each time period for small i.v. doses of conventional exogenous markers [3H-taurocholate, phenolphthalein glucuronide, indocyanine green, and horseradish peroxidase] and for less commonly studied endogenous markers [glucose, inorganic phosphate (Pi), total protein, and leucine aminopeptidase]. This temporal stability indicates a lack of confounding circadian variability for these markers during the course of the biliary function study. Biliary excretion patterns of these marker solutes (e.g., rapid high recoveries of phenolphthalein glucuronide and low concentrations of Pi and glucose) demonstrated that our system for bile function studies is associated with intactness of the examined pathways of bile formation. These results validate our system and set of marker solutes for in vivo biliary function studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Two cannula method for parenteral infusion and serial blood sampling in the freely moving rat

Materials and techniques are described for positioning and exteriorizing two chronic venous cannulas in the rat. A cannula for fluid infusion is placed in the inferior vena cava via the femoral vein and exteriorized on the tail, whereas a cannula for blood withdrawal is placed in the superior vena cava and exteriorized in the scapular region. Grooming behavior and range of motion are not impeded since the cannula used for chronic infusion is shielded by a light-weight tail cover instead of a torso harness. Application of this method can minimize numbers of rats used in total parenteral nutrition (TPN) studies as demonstrated by our feasibility study in which serum parameters of liver function were serially monitored in toxin-treated rats infused with nutrient mixtures.

Early effects of 1,1-dichloroethylene on canalicular and plasma membranes: ultrastructure and stereology

The pathogenesis of 1,1-dichloroethylene (1,1-DCE)-induced hepatotoxicity was investigated in fasted male rats by identifying the earliest morphological alterations in organelles. In situ perfusion-fixed liver tissue was examined by light and electron microscopy at 1, 2, or 3 hr after oral administration of 25, 50, and 100 mg 1,1-DCE/kg in mineral oil. The earliest morphological alterations, which occurred within 1 to 2 hr after 1,1-DCE administration, were dilation of bile canaliculi with an increase in the number of microvilli or membrane fragments in canaliculi and the formation of canalicular diverticuli in centrolobular hepatocytes. Subsequently, microvilli on sinusoidal surfaces were disrupted or lost. Membrane whorls were frequently found in bile canaliculi, the space of Disse, and between the lateral membranes of hepatocytes at early times. As injury progressed, centrolobular hepatocytes retracted from endothelial cells and sinusoidal plasma membranes invaginated to form cytoplasmic vacuoles. Stereological analysis of centrolobular hepatocytes at the 25 mg/kg dose showed a significant increase in canalicular volume density by 3 hr and no detectable alteration in mitochondrial volume density. These results indicate that changes in canalicular shape and microvilli configuration are the earliest morphological alterations following 1-DCE ingestion.

Vascular medial hyperplasia following chronic, intermittent exposure to 4,4′-methylenedianiline

Abstract4,4′-Methylenedianiline (DAPM) is an aromatic amine used in the synthesis of polyurethanes and epoxy resins. Acute exposure to DAPM produces hepatobiliary toxicity in humans as well as animal models. However, the toxic effects of intermittent DAPM exposure have not been explored. We treated male and female rats with 25 mg DAPM/kg or vehicle once per week for 17–22 wk. Though concentric fibrosis around bile ducts of the liver was noted, vascular medial hyperplasia was also prominent. Morphometric analysis of histologic sections revealed that in male rats, vessel wall area increased relative to lumen area in hepatic arteries by 22 wk. However, in female rats, wall areas of both hepatic and pulmonary arteries increased relative to lumen area by 17 wk. In both male and female rats, increased wall thickness was localized to the medial layer; no intimal changes were noted. In vitro treatment of vascular smooth muscle cells (VSMC) with 25–100 μM DAPM resulted in increased DNA synthesis and VSMC proliferation. To test whether the observed alterations in cell cycle control involved VSMC-mediated metabolism of DAPM to electrophilic intermediates, cells were treated with DAPM or DAPM plus 50 μM N-acetylcysteine (NAC). Coincubation with NAC afforded dramatic protection against DAPM-induced VSMC proliferation. Though DAPM had no appreciable effect on levels of reduced glutathione, oxidized glutathione, or oxidant production, DAPM increased glutathione-S-transferase activity in VSMC. These data indicate that DAPM can initiate VSMC proliferation, possibly via VSMC-mediated metabolism of DAPM to reactive intermediates.

Potentiation of 1,1-dichloroethylene hepatotoxicity: comparative effects of hyperthyroidism and fasting.

The responses of fed, fasted, and hyperthyroid (T4) Sprague-Dawley male rats to 50 mg 1,1-dichloroethylene (1,1-DCE)/kg were compared. Hyperthyroid rats received three sc injections of thyroxine (100 micrograms/100 g) at 48-hr intervals; all other rats were sham-injected. 1,1-DCE was given po in mineral oil 24 hr after the last T4 dose; controls received only mineral oil. Animals were killed at 2, 4, and 8 hr. Liver GSH contents were lowered about 55% by both fasting and T4 while GSH transferase activities were lowered about 20% by fasting and 35% by T4. Only T4 pretreatment lowered alcohol dehydrogenase activities. Liver injury (i.e., serum glutamate pyruvate transaminase, histology) after 1,1-DCE was minimal in fed rats, moderate in fasted rats, and intermediate in T4 rats. Fasted rats showed a more pronounced depletion of liver GSH after 1,1-DCE than T4 rats and only in fasted rats did the toxicant decrease activities of the detoxification enzymes. Hypoglycemia after 1,1-DCE occurred in fed rats, but more rapidly in T4 rats. In contrast, fasted rats unexpectedly became hyperglycemic after the toxicant. Patterns of body temperature change after the toxicant, which might be due to its metabolites, were dissimilar. Hypothermia was not observed in fed rats, was only transiently evident in T4 rats, but occurred rapidly within 1 hr in fasted rats and steadily became more severe. The dissimilar patterns of liver enzyme and body temperature and serum glucose change after the toxicant in the three groups are indicative of different pathways of injury potentiation by fasting and hyperthyroidism.

Biliary endogenous inorganic phosphate, D-glucose, IgA and transferrin are differentially altered by hydrostatic pressure

Our objective was to determine the effects of hydrostatic biliary pressure on excretion patterns of endogenous solutes which reflect various pathways of bile formation. A stable in vivo model was developed using anesthetized rats intraduodenally infused with taurocholate to maintain bile flow. Bile was collected during a 2-h basal period, a 4-h pressure period where elevation of the bile duct cannula decreased bile flow to 1/3 the basal rate, and a 2-h period after release of hydrostatic biliary pressure. During pressure treatment, bile salt concentration gradually increased approximately 3-fold, biliary inorganic phosphate concentrations rapidly rose approximately 5-fold, and biliary glucose concentration progressively rose approximately 17-fold. Concentrations of proteins in bile were affected differently with extreme decreases in IgA, moderate decreases in total protein and leucine aminopeptidase, and minimal change in transferrin. By 2 h after pressure release, only the alterations in biliary glucose and IgA persisted. The observed striking and persisting increases in biliary glucose are tentatively explained as an impaired reabsorption of glucose by the biliary tract.

Hyperthyroidism increases covalent binding and biliary excretion of 1,1-dichloroethylene in rats

Distribution, covalent binding, and biliary excretion of 1,1-dichloroethylene (DCE) were examined in euthyroid (EuT) and hyperthyroid (HyperT) rats, which are more vulnerable to DCE hepatotoxicity. Male Sprague-Dawley rats were made hyperthyroid by 3 sc injections of thyroxine at 48-h intervals prior to experiments; euthyroid controls received vehicle injections. A time course study monitored the circulation and excretion of 14C-DCE label for 24 h after administration of 14C-labeled DCE (50 mg/kg in mineral oil) in serial blood and urine samples. At 24 h, total and covalently bound 14C-label were measured in liver, kidney, and lung. Hepatotoxicity of DCE was enhanced in the HyperT rats, as evidenced by elevated serum activities of aminotransferase and histopathology, and was associated with increases in circulating metabolite, and in metabolite bound to red blood cells and liver but not to kidney or lung. Hyperthyroidism had little effect on in vitro capacity of hepatic microsomes to convert DCE to reactive intermediates as reflected by covalent binding. A biliary excretion study in pentobarbital-anesthetized rats showed a striking, but transient, increase in toxicant metabolite excretion in bile of HyperT rats during the first 2 h after toxicant administration (14C-DCE, 100 mg/kg). During the next 2 h, biliary metabolite excretion by HyperT rats decreased while there was a rise in circulating amounts of total and bound 14C-label. Thus, although hyperthyroidism had little effect on the total extent of DCE metabolized, this hormonal disturbance may have transiently enhanced metabolite formation and definitely was associated with a lesser ability to detoxify reactive DCE metabolites capable of injuring hepatic cell constituents by covalent binding reactions.

1,1-Dichloroethylene hepatotoxicity: hypothyroidism decreases metabolism and covalent binding but not injury in the rat.

Our objective was to determine if the previously reported protective effect of hypothyroidism against 1,1-dichloroethylene hepatotoxicity was associated with a change in distribution and covalent binding. Sprague-Dawley male rats were made hypothyroid (HypoT) by surgical thyroidectomy 2 weeks prior to studies and compared to euthyroid (EuT) rats. Hypothyroidism decreased body weights and liver to body weight ratios while mitochondrial non-protein sulfhydryl groups and cytosolic alcohol dehydrogenase activities were increased by 50%. Rats received a single oral dose of 100 mg [14C]1,1-dichloroethylene (DCE)/kg in mineral oil and were killed at 2, 4, 12 or 24 h; controls received mineral oil only. More rapid liver injury, as measured by serum alanine aminotransferase activity and histology, was present at 2 and 4 h after DCE in HypoT than EuT rats, but a similar magnitude of injury was evident at 12 and 24 h. DCE decreased liver non-protein sulfhydryl groups to a comparable extent in HypoT and EuT rats. Cytosolic glutathione S-transferase and alcohol dehydrogenase activities were decreased only in HypoT rats after DCE. HypoT rats excreted approximately 30% less total [14C]DCE-derived label in urine and their livers, kidneys and lungs consistently contained slightly less covalently bound [14C]DCE-derived label. In contrast, between 1 and 4 h after DCE, greater amounts of acid-soluble and acid-precipitable [14C]DCE-derived label were recovered in red blood cells of HypoT rats. Our results indicate that hypothyroidism did not protect against oral DCE hepatotoxicity but was associated with a more rapid injury at early times. Concurrently, hypothyroidism was found to change the fate of [14C]DCE with higher amounts of 14C-label recovered at early times in red blood cells while less 14C-label was excreted in urine and bound to liver.