Comparison of the efficacy of alpha-lactalbumin from equine, bovine, and human milk in the growth of intestinal IEC-6 cells.

Native alpha-lactalbumins (α-LA) from equine, bovine, and human milk were not cytotoxic. However, after treatment with trifluoroethanol (TFE), all three α-LAs exhibited cytotoxicity. Toxic potencies were distinctly different among them. Equine α-LA was the most robust, bovine α-LA was moderate, and human α-LA was weak. There were no significant structural changes as between the native and the TFE-treated α-LAs.

Trifluoroethanol increases albumin's susceptibility to chemical modification.

Acrolein-dependent chemical modification is implicated in the etiology of postoperative cognitive dysfunction (POCD). We examined this process further using human serum albumin (HSA), which is a target of acrolein modification and contains anesthetic binding sites. We tested whether trifluoroethanol (TFE), which mimics inhaled anesthetics, affects the susceptibility of HSA to modification by acrolein. We observed that acrolein promoted the formation of fluorescent adducts. TFE (10%) increased the amount of acrolein-HSA adducts. TFE (40%) caused a 5-fold increase in adduct formation. Acrolein also increased tryptophan anisotropy of HSA, which was further increased by TFE (10%). Acrolein-induced protein cross-linking was also increased in the presence of TFE (40%). These observations suggest that TFE promotes acrolein-induced modification of HSA, supporting a putative mechanism for POCD.

Sensory irritation mechanisms investigated from model compounds: trifluoroethanol, hexafluoroisopropanol and methyl hexafluoroisopropyl ether.

Quantitative structure-activity relationships (QSAR) have suggested the importance of hydrogen bonding in relation to activation of the sensory irritant receptor by nonreactive volatile organic chemicals. To investigate this possibility further, three model compounds with different hydrogen bond acidity, trifluoroethanol, hexafluoroisopropanol and methyl hexafluoroisopropyl ether, were selected for study. The potency of each chemical is obtained from the concentration necessary to reduce respiratory rate in mice by 50% (RD50). The RD50 values obtained were: methyl hexafluoroisopropyl ether (> or = 160,000 ppm), trifluoroethanol (11,400-23,300 ppm), and hexafluoroisopropanol (165 ppm). QSAR showed that trifluoroethanol and methyl hexafluoroisopropyl ether behaved as predicted as nonreactive sensory irritants, whereas hexafluoroisopropanol was much more potent than predicted. The higher than predicted potency of hexafluoroisopropanol could be due to a coupled reaction, involving both strong hydrogen bonding and weak Brönsted acidity. A concerted reaction could thus be more efficient in activation of the receptor. Hydrogen bonding properties and concerted reactions may be important in the activation of the sensory irritant receptor by nonreactive volatile organic chemicals.

Rat liver metabolism and toxicity of 2,2,2-trifluoroethanol.

2,2,2-Trifluoroethanol (TFE) is a metabolite of anesthetic agents and chlorofluorocarbon alternatives. Its toxicity in rats is a consequence of its metabolism to 2,2,2-trifluoroacetaldehyde (TFAld) and then to trifluoroacetic acid (TFAA). The enzymes involved in the toxic metabolic pathway have been investigated in this study. For the reaction of TFE to TFAld, the major hepatic metabolism associated with toxicity (as assessed by pyrazole-inhibitability) was NADPH dependent and occurred in the microsomes, whereas for TFAld conversion to TFAA, NADPH-dependent microsomal metabolism was significant, but mitochondrial and cytosolic metabolism in the presence of NADPH were also major contributors. NADPH-dependent hepatic microsomal metabolism of TFE to TFAld and TFAld to TFAA was inhibited by carbon monoxide, 2-allyl-2-isopropylacetamide, SKF-525A, metyrapone, imidazole, and pyrazole, and both reactions were oxygen dependent. The metabolism of TFE to TFAld was inhibited by diethyldithiocarbamate, a specific inhibitor of cytochrome P450E1, and by a monoclonal antibody to P4502E1, whereas the metabolism of TFAld was inhibited by neither. Ethanol pretreatment of rats enhanced the Vmax for hepatic microsomal metabolism of TFE to TFAld from 5.3 to 9.7 nmol/mg protein/min, while for TFAld to TFAA the Vmax was increased from 4.3 to 6.5 and the Km was unaffected for both reactions. Phenobarbital pretreatment of the rats did not affect any of these kinetic parameters. Coadministration of ethanol and a lethal dose of TFE very markedly decreased the lethality. Both the lethality (LD50 0.21 to 0.44 g/kg) and the metabolic kinetic parameters [(Vmax/Km)H(Vmax/Km)D = 4.2] were affected markedly when deuterated TFE replaced TFE. In contrast, deuteration of TFAld did not affect its lethality or rates of metabolism, but did affect its Km. Taken together these results indicate that P4502E1 catalyzed toxicity-associated hepatic metabolism of TFE to TFAld, while TFAld metabolism was catalyzed by a P450 which was not P4502E1. The hepatic metabolism of TFAld was not associated with its toxicity, which has been determined previously to be associated with its intestinal metabolism.

Trifluoroethanol and its oxidative metabolites: comparison of in vivo and in vitro effects in rat testis.

Trifluoroethanol (TFE) and trifluoroacetaldehyde (TFALD) produced a reduction in testis weight 3 days after a single oral dose of 10 mg/kg. In contrast, administration of trifluoroacetic acid (TFAA) caused no observable testicular effects. Reduction in testis weight was accompanied by morphological changes, involving specific damage to pachytene and dividing spermatocytes, and round spermatids. In an in vitro Sertoli/germ cell co-culture system, only TFALD was found to produce dose-related effects at concentrations of 10(-3) and 10(-4) M. There was increased germ cell loss from the cultures, particularly loss of pachytene and dividing spermatocytes, accompanied by leakage of the pachytene spermatocyte marker enzyme, lactate dehydrogenase-X. TFE and TFAA did not produce these effects in the culture system at concentrations equimolar with TFALD. These results suggest that TFALD may play a critical role in the development of the testis lesion seen with TFE in vivo. The effects seen both in vivo and in vitro were remarkably similar to those previously reported for another substituted alcohol and its metabolites, ethylene glycol monomethyl ether. It is postulated that the two series of compounds may have a similar mode of action on rat testis.

2,2,2-Trifluoroethanol toxicity in aged rats.

Various anesthetics are metabolized by different forms of cytochrome P-450 yielding the same toxic metabolite, 2,2,2-trifluoroethanol (TFE). The toxicity of TFE is a consequence of its metabolism catalyzed by cytochrome P-450. Since a marked age difference exists in the composition and inducibility of the hepatic mixed function oxidase system, we have elucidated the toxicity of TFE in 36-month-old aged Wistar rats. The aged rats were injected with sublethal doses of TFE (0.10 g/kg once per week for 5 weeks), after which they were sacrificed for pathologic examination. The major TFE-related lesions observed were severe hepatocyte degenerative changes such as basophilic, eosinophilic, vacuolated hepatocytes, bile duct hyperplasia, accumulation of lipofuscin pigments, and preneoplastic nodules. Other changes seen were the hyalinization of gastric submucosal wall, generalized testicular atrophy due to the loss of seminiferous tubules, coagulation necrosis of intestinal mucosal wall, hyperpigmentation, and more advanced and severe chronic progressive glomerulonephropathy in the TFE-treated rats. More severe lipofuscin and vacuolation of white matter of thalamic area, pons, midbrain, and cerebellum reflective of enhanced aging were also seen. Ultrastructural studies of liver from TFE-treated rats revealed rather diffuse loss of glycogen, fragmentation of endoplasmic reticulum, mineralization of mitochondria, and loss of other organelles within the hepatocytes versus saline-treated aged rats. Hepatic cytochrome P-450 concentrations, measured as a possible index of endoplasmic reticulum damage, were not affected by TFE administration.

Metabolism of 2,2,2-trifluoroethanol and its relationship to toxicity.

2,2,2-Trifluoroethanol (TFE), the toxic metabolite of the anesthetic agent fluoroxene, is further metabolized to trifluoroacetic acid, which accumulated to maximum serum concentrations 16 to 24 hr after TFE administration to rats. To determine how the metabolic pathways of TFE are related to its toxicity, male Wistar rats were pretreated with various metabolic inhibitors and inducers of cytochrome P-450 metabolism, and TFE toxicity and metabolism were assessed. Pyrazole, disulfiram, isoniazid, diethyldithiocarbamate, and 2-allyl-2-isopropyl-acetamide pretreatment significantly decreased the in vivo metabolism of TFE by 53-100% and decreased the toxicity (as assessed by mortality and leukocyte count alterations). Ethanol induction of rats increased metabolism of TFE by 65% 24 hr after TFE administration but not the toxicity. We conclude that hepatic ethanol-inducible cytochrome P-450 catalyzes the metabolism of most of the TFE but this metabolism is not associated with TFE toxicity, which probably arises by extrahepatic metabolism of TFE by another cytochrome P-450 form.

2,2,2-Trifluoroethanol-induced enteropathy in rats: chemically or bacterially mediated effects.

The lethal effects of the fluorinated ether anesthetic, fluroxene, in rats are a consequence of its metabolism, which is catalyzed by cytochrome P-450 to the toxic metabolite 2,2,2-trifluoroethanol (TFE). The anesthetic or TFE (0.21 g/kg) caused decreased white blood cell counts, necrosis of bone marrow and lymphocytes, and decreased small intestine dry weight and was associated with septicemia. To elucidate the mechanism of TFE toxicity in rats we undertook histopathologic, ultrastructural and bacteriologic studies. TFE produced severe edema of intestinal lamina propria and submucosae, dilatation of crypts, loss of surface epithelium, vacuolation and necrosis of intestinal epithelial cells, and infiltration of polymorphonuclear leukocytes into the edematous lamina propria. Intestinal epithelial villi lost their cellular tissue integrity. Coccobacillary organisms were numerous in the ulcerated intestine. Hemolytic E. coli were isolated from intestinal tissue at a two-log increase in concentration relative to controls. Hemograms from TFE-treated rats exhibited marked leukopenia and morphologic differences. The platelets lost their discoid shape, extended pseudopods, and centralizing granules. Hemoglobin precipitation as Heinz bodies and crystalloid structures was observed in TFE-treated rats. Together the data suggest that TFE-induced enteropathy was most probably due to E. coli precipitated from TFE-mediated alterations in the population of small intestinal microbes. The antibiotics erythromycin, active against gram-positive bacteria, and streptomycin, active against gram-negative bacteria, and the antiendotoxin, polymyxin B, were administered to rats prior to TFE in an effort to differentiate between these mechanisms by altering the intestinal bacteria populations. The results indicate that the TFE-induced small intestinal lesions are initiated by the direct focal necrotic effect of TFE or its metabolites on the small intestinal epithelium. The focal coagulation necrosis produced by TFE predisposes the animals to lethal enteritis and systemic bacteremia.

2,2,2-Trifluoroethanol toxicity in hamsters (Mesocricetus auratus).

As part of an effort to further elucidate the mechanism whereby fluorinated ether anesthetics express their various toxic effects, Golden Syrian hamsters were utilized to determine acute and subchronic toxicities of the anesthetic metabolite 2,2,2-trifluoroethanol (TFE). The major lesion observed with the TFE (0.25 g/kg ip) was coagulation necrosis characterized by nuclear pyknosis in epithelial cells of intestinal villi, the deeper portion of gastric mucosae, lymphoid organs such as thymus, spleen and mesenteric lymph nodules and brain. Hepatocyte hypertrophy with diffuse vacuolar degeneration was also found. Lungs had a characteristic focal terminal bronchiolar epithelial hyperplasia with discrete mucous metaplasia in the terminal bronchioles. In subchronic studies, hamsters were injected with sublethal doses of TFE (0.20 g/kg once per week for 5 weeks). The major lesions observed were severe hepatocyte hyperplasia and hypertrophy with fatty degeneration, necrosis of hair germinal matrix, urinary bladder transitional epithelial cellular hypertrophy, and hyperplasia with vacuolar degeneration and submucosal edema. Ultrastructural studies of acutely treated animals indicate that fat vacuoles formed within the hepatic endoplasmic reticulum characterized by lamellar bodies forming multiple myelin figures within the fat vacuoles. Hepatic cytochrome P-450 concentrations measured as a possible index of endoplasmic reticulum damage, failed to reveal any significant differences between the treated and non-treated hamsters.

Mechanism of toxicity of 2,2,2-trifluoroethanol in rats.

2,2,2-Trifluoroethanol (TFE) is the toxic metabolite of the anesthetic agent fluroxene. TFE treatment (0.21 g/kg, ip) of male Wistar rats significantly reduced peripheral white blood cell count, bone marrow nucleated cellularity, and dry weight of the small intestine. These toxic effects of TFE were first observed at 8 to 16 hr after treatment, persisted for 96 hr, and were accompanied by severe diarrhea and edema of the small intestine. A non-lethal dose of TFE increased the sensitivity of rats to bacterial endotoxin lethality by approximately 1000-fold. Antibiotic and antiendotoxin pretreatment reduced the lethality of TFE from 80 to 20% of the rats, but did not prevent the other toxic effects of TFE. In vitro experiments with serum from TFE-pretreated rats (0.13 g/kg) supported the growth of an average of 65% fewer cultured bone marrow cell colonies compared to the number of colonies produced when serum from control rats was used. This suggests that TFE-induced bone marrow depression and leukopenia are related to a decrease in colony stimulating factor activity. Taken together these results explain the rapid development of lethal bacterial infections in TFE-treated rats. TFE-mediated damage to the small intestine combined with prolonged leukopenia decreases the resistance of the rat to endogenous pathogens leading to systemic bacterial infection. In addition, the increased sensitivity to endotoxin induced by TFE leads to lethal endotoxemia.

Trifluorinated ether anesthetic lethality in rats: the role of bacterial infection.

The lethal effects of the fluorinated ether anesthetics fluroxene (2,2,2-trifluoroethyl vinyl ether) and its ethyl (TFEE) and allyl analogues in male Wistar rats have previously been demonstrated to be potentiated by specific hepatic microsomal cytochromes P-450, and mediated by the common metabolite 2,2,2-trifluoroethanol (TFE). We report here that administration of lethal combinations of anesthetic and cytochrome P-450-inducing agents or of lethal doses of TFE (0.21 g/kg and higher) to rats caused decreased white blood cell counts, necrosis of sternum bone marrow cells and lymphocytes in the thymic cortex, and resulted in Escherichia coli contamination of the blood, lungs, liver, and kidneys of treated rats. Control animals in identical environments were free of bacterial contamination. Pretreatment of rats with the antibiotic tetracycline-HCl in the drinking water (0.6 g/liter) from 24 hr before anesthetic or TFE administration significantly diminished the mortality. With TFEE and beta-naphthoflavone induction, mortality was reduced from 85 to 30% by the antibiotic. However, the antibody plaque assay following immunization with sheep erythrocytes indicated that the primary humoral immune response to a thymus-dependent antigen was not impaired in treated rats. These results considered together indicate that metabolic formation of TFE from the anesthetic agents produced a decreased host resistance with subsequent increased susceptibility to bacterial infection. If not administered the antibiotic, the animals succumbed to the infection.

Absence of mutagenic activity of trifluoroethanol and its metabolites in Salmonella typhimurium.

Trifluoroethanol, trifluoroacetaldehyde and trifluoroacetate were found to have no mutagenic activity in the standard Salmonella typhimurium reverse mutation assay (Ames test) using a closed incubation system. Negative results were also obtained when incubation mixtures included 9000 x g supernatant fractions of rat liver or testes homogenates along with an NADPH generating system. Rats were pretreated with a polychlorinated biphenyl mixture to induce biotransforming enzyme activity. These results suggest that the previously reported mutagenic activity of fluroxene is not due to metabolites arising from the trifluoroethyl side of the molecule and that inhibition of spermatogenesis in rats by trifluoroethanol is not mediated through a mutagenic mechanism.

Ocular toxicity of 2,2,2-trifluoroethanol in rabbits.

Three commercial formulations of 2,2,2-trifluoroethanol (TFE) were applied to eyes of rabbits and evaluated for their potential to cause ocular damage. All three products were severe eye irritants and water irrigation shortly after exposure was not effective in reducing ocular damage. Gross and histological observations revealed marked conjunctivitis, moderate to severe ulcerative keratitis, and hyperemia and edema of the iris, all of which showed evidence of resolution over the three-week course of investigation. However, complete reversal of all effects was not attained. TFE and its commercial formulations should be viewed as potentially severe human ocular irritants.

Sex differences in anaesthetic toxicity: fluroxene and trifluoroethanol in mice.

A sex difference in postanaesthetic mortality after fluroxene anaesthesia was found in Swiss Webster mice. More males succumbed than females. This toxicity was biotransformation-dependent and could be reversed by pretreatment with "opposite" sex hormones. The toxicity of the fluroxene metabolite trifluoroethanol also was more marked in male mice, but was only partially influenced by microsomal enzyme inhibitors or stimulators, or by sex hormones.