Effect of CYP2E1 induction by ethanol on the immunotoxicity and genotoxicity of extended low-level benzene exposure.

Potential additive effects of ethanol consumption, a common life-style factor, and low-level benzene exposure, a ubiquitous environmental pollutant, were investigated. Ethanol is a potent inducer of the cytochrome P-450 2E1 (CYP2E1) enzyme, which bioactivates benzene to metabolites with known genotoxicity and immunotoxicity. A liquid diet containing 4.1% ethanol was used to induce hepatic CYP2E1 activity by 4-fold in female CD-1 mice. Groups of ethanol-treated or pair-fed control mice were exposed to benzene or filtered air in inhalation chambers for 7 h/d, 5 d/wk for 6 or 11 wk. The initial experiment focused on immunotoxicity endpoints based on literature reports that ethanol enhances high-dose benzene effects on spleen, thymus, and bone marrow cellularity and on peripheral red blood cell (RBC) and white blood cell (WBC) counts. No statistically significant alterations were found in spleen lymphocyte cellularity, subtype profile, or function (mitogen-induced proliferation, cytokine production, or natural killer cell lytic activity) after 6 wk of ethanol diet, 0.44 ppm benzene exposure, or both. This observed absence of immunomodulation by ethanol alone, a potential confounding factor, further validates our previously established murine model of sustained CYP2E1 induction by dietary ethanol. Subsequent experiments involved a 10-fold higher benzene level for a longer time of 11 wk and focused on genotoxic endpoints in known target tissues. Bone marrow and spleen cells were evaluated for DNA-protein cross-links, a sensitive transient index of genetic damage, and spleen lymphocytes were monitored for hprt-mutant frequency, a biomarker of cumulative genetic insult. No treatment-associated changes in either genotoxic endpoint were detected in animals exposed to 4.4 ppm benzene for 6 or 11 wk with or without coexposure to ethanol. Thus, our observations suggest an absence of genetic toxicity in CD-1 mice exposed to environmentally relevant levels of benzene with or without CYP2E1 induction.

Decreased biliary secretion of proteins and phospholipids by rats with 1,1-dichloroethylene-induced bile canalicular injury.

1,1-Dichloroethylene (DCE, 50 mg/kg) rapidly and selectively injures the bile canalicular membrane of zone 3 hepatocytes. Thus, DCE is of value as a tool to assess the consequences of alterations in canalicular membrane integrity on bile formation. Our objective was to characterize the effects of DCE on the biliary secretion of proteins and phospholipids in freely moving rats. DCE treatment caused a rapid and sustained decrease in total biliary protein output. In contrast, canalicular membrane-localized enzyme activities more slowly increased to 8- to 15-fold in bile from DCE-treated rats. Biliary output of lysosomal enzymes was altered in a biphasic manner. Specifically, there was a transient fivefold increase within 30 min of DCE treatment and then a progressive decrease to approximately 10% basal levels by 4 h. Secretion of phospholipids into bile decreased rapidly in a striking and sustained manner, after DCE. Our findings of diminished lysosomal protein and phospholipid secretion following DCE treatment are consistent with an important role for canalicular membrane integrity in their entry into bile.

Repeated oral benzene exposure alters enzymes involved in benzene metabolism.

Benzene is a known carcinogen and hematopoietic toxin in humans and experimental animals. The effect of acute, high-dose exposure to benzene on hepatic bioactivation and detoxication enzymes has been defined, while little is known about the effect of repeated, low-dose benzene exposure on these enzymes. Our objective was to determine whether repeated, oral benzene exposure alters enzymes involved in benzene metabolism. Specifically, we were concerned with cytochrome P-450-2E1, a bioactivation enzyme, and glutathione transferase and aldehyde dehydrogenase, two detoxifying enzymes. Female CD-1 mice were treated by gavage for 3 wk with benzene doses of 5 mg/kg (0.064 mmol/kg) or 50 mg/kg (0.646 mmol/kg) in corn oil. These doses of benzene produced 0.048 and 0.236 mumol muconic acid/d, respectively. We found that repeated exposure to 50 mg benzene/kg/d decreased P-450-2E1 activity by 34% and induced glutathione transferase activity by 30% without affecting aldehyde dehydrogenase activity. These changes in enzyme activities may serve a protective role against repeated exposure to benzene.

Calcium channel blockers induce thymic apoptosis in vivo in rats.

We investigated the in vivo effect of structurally different calcium channel blockers (CCB) on rat thymus. Administration of verapamil (40 mg/kg ip), diltiazem (90 mg/kg ip), nifedipine (15 mg/kg ip), or nicardipine (10 mg/kg ip) induced apoptotic indices of 4.3, 4.0, 2.0, and 6.5, respectively, compared to 0.5 in the saline-treated control rats. Apoptosis was assessed by morphology and the apoptotic index was calculated using a computer-assisted image analyzer. Diltiazem had a rapid and substantial effect as evidenced by apoptosis at 1.5 hr and a 36% decrease in thymus weight by 24 hr. We were uncertain about the mechanisms by which CCB induced thymic apoptosis in vivo since in vitro studies have shown that increases in intracellular calcium cause apoptosis and that CCB prevent apoptosis. We sought insight into the mechanism by evaluating potential and known in vivo effects of these drugs. Neither verapamil nor diltiazem was found to elevate serum cortisol levels, a known trigger for apoptosis. Hypotension, a known response to CCB, does not appear to be causal factor since the potent hypotensive agent sodium nitroprusside (10 microg/kg, iv) did not cause a significant increase in thymic apoptosis. Calcium signaling may be important since the calmodulin antagonist chlorpromazine (60 mg/kg ip) was found to induce a 15-fold increase in apoptosis. Our observations suggest that calcium signaling is necessary for the survival of the T lymphocytes in the thymus.

Species differences in testicular and hepatic biotransformation of 2-methoxyethanol.

Biotransformation of 2-methoxyethanol (2-ME) by alcohol and aldehyde dehydrogenases is an established factor in the toxicity of this useful solvent. Little is known about potential capacity for 2-ME biotransformation by testis or other target tissues. We detected appreciable capacity for 2-ME biotransformation by alcohol dehydrogenase in testes from Sprague-Dawley rats. However, kinetic analysis showed a 6-fold lower affinity for 2-ME by alcohol dehydrogenase of testis compared to liver. 2-ME biotransformation was also detected in testes from Wistar rats and one strain of mice but not in testes from hamsters, guinea pigs, rabbits, dogs, cats or humans. Testes from all these species readily converted the aldehyde metabolite of 2-ME to 2-methoxyacetic acid. Hepatic capacities for 2-ME biotransformation by alcohol dehydrogenase varied from 22 to 2.5 mumol/mg prot/min with a species rank order of: hamsters >> rats = mice > guinea pigs = rabbits. There was no consistent concordance between activities for 2-ME versus ethanol, the prototype substrate for alcohol dehydrogenase, which could reflect substrate preferences of different isozymes. Species differences between rats and hamsters were also found for testicular and hepatic biotransformation of the glycol ethers, 2-ethoxyethanol and 2-butoxyethanol. Although species differences in capacity for 2-ME biotransformation were found, the observations do not provide an explanation for reported species and strain differences in susceptibility to 2-ME toxicity.

Apoptosis: molecular control point in toxicity.

Apoptosis is a controlled form of cell death that serves as a molecular point of regulation for biological processes. Cell selection by apoptosis occurs during normal physiological functions as well as toxicities and diseases. Apoptosis is the counterpart and counterbalance to mitosis in cell population determination. Complex patterns of cell signaling and specific gene expression are clearly involved in the control of cell fate. Exposure to an apogen, a trigger of apoptosis, can significantly increase apoptotic cell loss during homeostatic processes as well as acute or chronic toxicities. Alternately, suppression of apoptosis through, for example, interference in cell signaling can result in pathological accumulation of aberrant cells and diseases such as tumors. Investigations into the mechanisms underlying apoptosis have extended into many areas, driven by increasingly sophisticated instrumental and molecular biology techniques. This symposium summary explores related aspects of apoptosis, including control of cell population size and function, specific gene activity and regulation, chromatin condensation and scaffold detachment, oxidative stress-induced cell proliferation versus death by apoptosis or necrosis, and hepatotoxicant-induced apoptosis versus necrosis. Insights into the mechanisms governing apoptosis and increasing appreciation of the relevance of apoptotic cell death are redirecting research in toxicology and carcinogenesis and are yielding novel therapeutic approaches for the control of toxicity, disease, and ultimately perhaps senescence.

Biliary glutathione and some amino acids are markedly diminished when biliary pressure is elevated.

We studied the effects of a transient elevation in biliary pressure on biliary glutathione and amino acids in rats. Other biliary solutes monitored were total bile salt, Pi, which is a putative marker of paracellular leakage, and glucose, which is reabsorbed from the biliary tract. Experiments were carried out on anesthetized rats intraduodenally infused with taurocholate to maintain bile flow during a 2-hr basal period, a 4-hr pressure period during which the bile duct cannula was elevated until bile flow decreased to 1/3 the basal rate, and a 2-hr period after release of hydrostatic biliary pressure. We found that pressure treatment caused biliary concentrations of glutathione to progressively decrease by 80%, while biliary Pi rapidly rose approximately 3- to 4-fold, bile salt gradually increased approximately 3-fold, and biliary glucose concentration progressively rose 15-fold. HPLC analysis of monobromobimane-derivatized biliary thiols indicated that the decline in biliary glutathione was not accompanied by an increase in its breakdown products, cysteine and cysteinylglycine. Pressure treatment led to four patterns of change in biliary amino acid concentrations: (1) increases of 29 to 76% for the basic amino acids lysine and arginine, which have very low bile/plasma ratios of about 0.1; (2) no change for the more water soluble amino acids with bile/plasma ratios close to 1.0, e.g., histidine and urea; (3) modest decreases of 16 to 48% for a variety of amino acids including serine, glutamate, and glycine; and (4) marked, progressive decreases of > 50% for aromatic and branched chain amino acids. By 2 hr after release of pressure, only the alterations in biliary glucose and some amino acids, particularly the branched chains, persisted. This is the first report of cholestasis-induced alterations in biliary amino acids.

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.

Reactive oxygen species in normal physiology, cell injury and phagocytosis.

Formation of free radicals and other ROS is a continuous aspect of life. Examples include the free radical intermediates which are formed by the nonenzymatic peroxidation of polyunsaturated fatty acids of membrane lipids in a destructive process and which are also formed by the enzymatic peroxidation of arachidonic acid in the biosynthesis of potent chemoattractants. Organisms cope with these reactive species by a variety of strategies that limit formation of ROS or remove cytotoxic products. Oxidative burst reactions that yield ROS provide an effective, vital process for killing invading organisms. Research on why ROS formation is impaired in phagocytic cells of people with chronic granulomatous disease has provided new insights into the complexity of the factors that prevent inadvertent activation of this destructive force.

Biliary excretion of marker solutes by rats with 1,1-dichloroethylene-induced bile canalicular injury.

Our objective was to characterize the effects of 1,1-dichloroethylene (DCE, 50 mg/kg) on bile formation by freely moving rats. This toxicant provides a new tool to study structure-function associations because it selectively injures zone 3 bile canaliculi of fed rats. Ultrastructural changes included loss of microvilli from the canalicular membrane and membrane fragments within the dilated canalicular lumens. Function studies compared biliary excretion of a battery of endogenous and exogenous marker solutes during a basal period versus that at 1 and 3 hr after toxicant treatment. DCE treatment had the following effects on biliary functions in fed rats: a slowing of organic anion transport into bile; a decrease in biliary total protein content; a striking increase in biliary leucine aminopeptidase, a canalicular membrane protein; and an accentuation of the early entry of horseradish peroxidase into bile without a change in the total amount of this large protein recovered in bile. In contrast, bile flow and bile salt excretion were not altered, Pi continued to be excluded from bile, and D-glucose continued to be reabsorbed from bile. The observed alterations in biliary functions and canalicular structure may ensue from the metabolism of DCE to semistable glutathione conjugates that congregate near the canaliculi en route to biliary excretion.

Increased biliary transferrin excretion following parenteral aluminium administration to rats.

Aluminium accumulates in the livers of patients receiving either parenteral nutrition or haemodialysis. When given parenterally to rats, aluminium causes cholestasis. However, the mechanism of hepatic aluminium uptake and the fate of aluminum in the liver are poorly understood. We examined the effect of parenteral aluminium administration on biliary excretion of transferrin, the major circulating aluminum-binding protein. Male Wistar rats were given parenterally aluminum 5 mg/kg/day for 1-14 days. Bile was collected for 3 hr at the end of the study period. Biliary total protein concentration and IgA/total protein were unaffected by up to 14 days of parenteral aluminium administration. However, biliary transferrin excretion increased with duration of aluminum administration up to five-fold by day 14. Biliary transferrin concentration and transferrin/total protein was higher in aluminum treated rats than controls after 7 and 14 days of study. Hepatic aluminum concentration reached a maximum after 4 days of parenteral aluminum administration, at which time serum bile acid and alanine amino transferase values were not different from controls. Since biliary transferrin is normally derived from the serum, it is likely that aluminum promotes hepatocellular uptake of transferrin and that aluminum enters the hepatocyte bound to transferrin. We postulate that transferrin may direct aluminum to intracellular sites where its toxic effects would be minimized.

Total parenteral nutrition-associated alterations in hepatobiliary function and histology in rats: is light exposure a clue?

Light exposure of amino acids, especially in the presence of photosensitizers such as riboflavin, has been shown to result in photoproducts that are toxic both in vivo and in vitro. Provision of photooxidized single amino acids has been shown to result in hepatic dysfunction in both gerbils and rats. However, studies of the effects of light exposure of complete nutrient mixtures (glucose-amino acid-vitamins) on hepatobiliary responses are limited. These are important because, in clinical practice, total parenteral nutrition (TPN) solutions are continually exposed to light and because hepatic dysfunction is the most common metabolic aberration associated with TPN. Studies were conducted to compare the effects of TPN that had been exposed to light (+L) or protected from light (-L) on hepatobiliary function of rats. TPN solutions were either exposed to light or protected from light for 24 h and then infused into rats for 10 d. Data for enterally fed rats are also shown for comparison with parenterally fed animals. +L animals lost more weight and had lower bile flow, higher taurocholate output in bile, higher biliary osmolarity, and higher inorganic phosphate in bile. Hepatic histology demonstrated scattered foci of necrosis in eight of the eight +L animals and in only one of eight -L animals. These data demonstrate that protection of TPN solutions from light minimizes TPN-associated alterations in hepatobiliary function and histology. These histologic changes observed in the +L animals are in contrast to previous reports of TPN-induced histologic changes, suggesting a different mechanism. Our observations suggest that consideration be given to the implications of exposing TPN solutions to light.

Hepatic function during short-term total parenteral nutrition: effect of exposure of parenteral nutrients to light.

Total parenteral nutrition (TPN) solutions either exposed to light (+L) or protected from light (-L) were infused for 5 days through jugular cannulas in freely moving rats placed in metabolic cages. At the end of the 5 day period, bile flow, biliary inorganic phosphate and biliary gamma-glutamyl transferase activity, as well as biliary concentrations of several essential and branch chain amino acids were significantly lower in the -L animals compared to +L animals. In addition, biliary glutathione was significantly lower in the +L animals. In both groups of animals, plasma tyrosine decreased significantly from pre-TPN values despite a doubling of plasma phenylalanine concentrations suggesting that tyrosine may become a conditionally essential amino acid in rats provided TPN. Our findings indicate that short-term parenteral infusion of light exposed TPN solutions alters hepatobiliary function as well as amino acid homeostasis and that the changes are minimized by light protection of the infusates. The exact mechanisms of the contribution of light exposure in the induction of hepatic dysfunction remain to elucidated.

Glutathione and tissue amino acid responses to light-exposed parenteral nutrients.

Effects of infusion of light-exposed (+L) or light-protected (-L) total parenteral nutrition solutions were investigated in rats. The parenteral infusions were carried out for 7 days through jugular cannulas in freely moving rats in metabolic cages. Plasma tyrosine and citrulline, hepatic methionine, valine, isoleucine, leucine and tyrosine, and biliary cystathionine were significantly greater in the -L than +L rats, whereas biliary arginine was significantly lower in the -L compared to +L rats. Bile flow, biliary inorganic phosphate and glucose were significantly lower, whereas biliary total glutathione (GSH+GSSG) was significantly greater in the -L compared to +L animals. These data suggest adverse effects on hepatobiliary function due to light exposure of parenteral nutrients. The endogenous markers used suggest that tight junction permeability, bile acid-independent flow, glutathione and amino acid homeostasis are altered by light exposure and that these changes can be minimized by light protection. The mechanisms involved in the induction of these changes need to be elucidated. The role of light exposure of parenteral nutrients during routine clinical use in the induction of hepatic dysfunction, a common metabolic complication of parenteral nutrition, needs to be considered.

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.

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 rat's 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)

Identification of an anion-transport ATPase that catalyzes glutathione conjugate-dependent ATP hydrolysis in canalicular plasma membranes from normal rats and rats with conjugated hyperbilirubinemia (GY mutant).

Rat liver canalicular plasma membranes were found to contain a 37-kDa protein that is immunologically cross-reactive with the dinitrophenyl glutathione-stimulated ATPase previously identified in human tissues. The protein, which was partially purified by affinity chromatography, exhibited ATPase activity dependent on dinitrophenyl glutathione, bilirubin ditaurate, and other dianionic compounds. The localization of this protein in the canalicular membrane and its measured enzymatic activity indicate that it is involved in the transport of glutathione derivatives and other dianionic organic compounds. A rat mutant in which the above transport activities are impaired contained the protein in amounts similar to those in a normal control.

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.