Role of biotransformation in the toxicity of inhalation anesthetics

Life Sciences, Vol . 23, pp . 2447-2462 Printed in the U .S .A .

Pergamon Press

MINIREVIEW ROLE OF BIOTRANSFORMATION IN THE TOXICITY OF INHALATION ANESTHETICS Robert W . Yaughan, I . Glenn Sipes and Burnell R . Brown, Jr . Department of Anesthesiology and Toxicology Program, University of Arizona, College of Medicine, Tucson, Arizona 85724

Role of Biotransformation in Inhalation Anesthetic Toxicity Volatile liquids were first employed as anesthetics 130 years ago . It was apparent that these volatile inhalation anesthetics possessed properties of powerful protoplasmic poisons . They could retard or destroy biologic functions at all levels from subcellular . organelles to whole organisms . Most familiar to anesthetists today are the acute effects of anesthetics on cardiopulmonary function . As stated by Cascorbi (1), depression of biologic function by anesthetics is dose dependent and reversible . Subacute (viscerotoxic) and chronic (trace concentration) toxicity of volatile anesthetics are different problert~ . Cascorbi (1), defined subacute toxicity of anesthetics as organ damage occuring after the central nervous system effects of general anesthesia have disappated, usually within days after anesthesia . Most commonly affected are the organs of drug metabolism (ie . biotransformation) and excretion, eg . the liver and kidney . Halothane has been implicated in postoperative hepatic damage (2), methoxyflurane in nephrotoxicity (3), and questions are being raised about the possible nephrotoxicity of enflurane (4,5) . This review will focus on subacute and chronic viscerotoxicity due to inhalation anesthetics . The anesthetic agents considered are chloroform, trichloroethylene, fluroxene, methoxyflurane, halothane, enflurane, and isoflurane . BIOTRANSFORMATION As recently as ten years ago it was a dictum among anesthesiologists and pharmacologists that clinically employed inhalation anesthetic gases and vapors were metabolically inert (6) . Trichloroethylene was recognized as an exception since this anesthetic was known to be extensively metabolized (7,8) . However, this observation was considered of little more than academic interest for several decades . From the point of view of the practicing anesthetist, there are two possible implications to anesthetic metabolism : 1)influence on the conduct of the anesthetic 2) association with organ toxicity (6) . The likelihood that metabolism might alter anesthetic requirement is remote since inhalation anesthetics are administered in an amount far in excess of utilization . However, if biodegradation proceeds to biologically active or toxic compounds, anesthetic metabolism becomes a matter for serious concern . During metabolism xenobiotics undergo physiochemical changes that alter their biological properties (9) . As a general rule, lipophilic, nôn-polar compounds (which include the majority of anesthetics and hypnotics) are con verted into polar, water-soluble derivatives capable of being excreted via the urine and bile . Non-volatile (fixed) drugs are excreted only by the liver and kidney, with no egress via the lungs . Lipophilic drugs, if unchanged, pass through the lipid membranes of the renal tubular epithelial cells following glomerular filtration and reenter the systemic circulation . Thus, without 0300-9653/78/1218-244702 .00/0 Copyright (c) 1978 Pergamon Press

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hepatic biotransformation forming polar derivatives, compounds taken into the body may remain there for prolonged periods . For example, it has been estimated that the half-life of the ultrashort acting barbiturates, thiopental, would be approximately 20 years were it not for metabolic conversion (6) . During metabolism most drugs undergo detoxification, ie ., conversion to derivatives less pharmacologically active and theoretically less toxic than However, there are specific examples where biotransfor the parent compound . mation results in the formation of metabolites with greater toxicity than the parent compound . Two well studied examples include bromobenzene and acetaminophen, both biodegraded to metabolites of marked hepatotoxicity (10,11) . Mary halogenated alkanes and alkenes are biotransformed to reactive intermediates or toxic metabolites . Since most modern inhalation anesthetics belong to this class, concern about bioactivation and toxicity is of clinical as well as occBecause of its historical significance and well documented upational concern . hepatotoxic actions, this review on inhalation anesthetics will begin with chloroform . Chloroform Besides its use as an anesthetic, chloroform (CHC13) as well as the alkyl halide, carbon tetrachloride (CC14) are widely used solvents . These agents are known to be hepatotoxic, and they are also suspected carcinogens . The hepatotoxicity of these drugs has been attributed to the interaction of highly reactive intermediates of biotransformation (ie . free radicals) with tissue macromolecules . This interaction results in an alteration of cellular It is generally assumed that these integrity and tissue necrosis (12-15) . active intermediates are produced by oxidative hepatic microsomal enzymes (16-20) . Scholler (21) demonstrated that increasing the rate of chloroform metabolism by hepatic microsomal enzyme induction with phenobarbital enhanced hepatic necrosis produced by chloroform, whereas inhibiting biotransformation or supplying antioxidants reduced the severity of the necrosis . Normally, the liver possesses antioxidants or free radical scavengers which protect important structural or biochemical lipids and proteins from interaction with active However, if the liver is exposed to very high concenintermediates (22,23) . tration of active intermediates (eg . enzyme induction), or if the level of endogenous protective agents is reduced, tissue destruction may occur . (GSH), a sulfhydryl Recent findings indicate that reduced glutathione containing tripeptide found in high concentrations in the liver, may play a key role in protecting the liver from the toxic effects of bromobenzene (10), acetaminophen (11), and carbon tetrachloride (24) . The actual mechanism by which GSH reduces tissue damage is~not known, but it is suggested that the covalent binding of the active intermediates of these cômpounds to tissue macromolecules is reduced (11,25,26) . Also it appears that the microsomal lipid peroxidation induced by CC14 is prevented by GSH (27) . Chloroform anesthesia has been abandoned because of the sporadic occurrence of hepatic necrosis associated with this agent . The mechanism of chloroform liver damage is now fairly clear and can serve as a model for hepatotoxicity . The metabolic fate of chloroform has been extensively studied in animals, but only limited data are available in humans . Fry and co-workers (28) have used the sophisticated technique of isotope ratio-mass spectrometry to examine metabolism of chloroform in two normal subjects following oral administration of gelatin capsules containing 500mg of chloroform 13 C dissolved in olive oil . The per cent of administered chloroform 13 C recovered as 13C02 was 48 .5 and 50 .6 . There appeared to be no evidence for other exhaled metabolites, although these were carefully searched In various animals studied chloroform is metabofor by gas chromatography . lized to CO2, chloride ion, and glutathione-conjugated chloromethyl derivatives . These urinary metabolites are innocuous . However, during biotransfor-

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mation of chloroform and conversion to these final products, free radicals or reactive intermediates are produced capable of destroying liver cell integrity by the process of oxidative destruction of unsaturated lipids (lipoperoxidation) . Unsaturated lipids are keystones of intracellular membrane structure . Ordinarily, this lipoperoxidation process is quenched by antioxidants, such as reduced glutathione normally present in the cell, and the reaction never reaches sufficient intensity to cause necrosis of the liver . Brown and colleagues (29) studied the effects of hepatic microsomal enzyme induction with phenobarbital in rats on the acute hepatotoxic responses to chloroform . Phenobarbital pretreatment markedly increased the hepatotoxic response to chloroform anesthesia . Hepatic GSH levels were decreased 70-80~ after 2 hours of chloroform anesthesia in induced rats, but were unchanged in non-induced rats in which no light microscopic evidence of toxicity developed . Marked destruction of microsomal electron transfer components were observed in the chloroform-anesthetized, phenobarbital induced animals only . Depletion of liver GSH by pretreatment of rats with diethylmaleate markedly enhanced CHC13 induced hepatotoxicity . Microsomal induction caused a largé increase in in-vitro covalent binding of 1 ~CHC13 metabolites to microsomal protein, which coü~be d prevented by GSH . Hepatotoxicity of chloroform appears to be related to two factors : (1) rate of biotransformation ; (2) availability of the hepatic antioxidant, GSH . Therefore, it is the condition of the liver that determines chloroform hepatotoxicity . The vector seems to reside with biotransformatiorl of the anesthetic (29) . Trichloroethylene (TCE) Although trichloroethylene has limited clinical usefulness, its metabolism is of interest in that it was the first of the inhalation anesthetics shown to be biotransformed (7,8) . In addition, a wide industrial exposure to this agent continues . Work of Powell (7) and Soucek and Vlachova (30) have shown the primary metabolic products to be trichloroethanol and trichloroacetic acid (TCS), the same products as found in animal studies . This conversion is unique since it was found that it took place not by the loss of chloride but by a rearrangement of the chlorines on the carbon skeleton (31) . The possibility that chloral hydrate is an intermediate in the metabolism of trichloroethylene was proposed by Butler (8), but only recently was this proven to be the case . Leibman (32) was able to isolate chloral hydrate after incubation of microsomas with trichloroethylene and also found this metabolism In man TCA may be detected in the to be inducible with phenobarbital (33) . urine for as long as 48 hours following TCE anesthesia . Recently, TCE has been shown to induce hepatocellular carcinoma in mice (34) . A number of expermental studies have provided evidence that some carcinogens are metabolized to activated carcinogenic intermediates by means of cytochrome P-450 drug metabolizing enzymes (35, 36) . The intermediate epoxides from such carcinogens may then interact covalently at nucleophilic sites in nucleic acids and proteins . One or more of these processes are probabl responsible for their carcinogenic activity (37) . Van Duren and Banerjee (37~ examined the covalent interaction of TCE metabolites with rat liver microrosomas in vitro . Their results showed that TCE binds covalently to microsomal prote~ since extensive extractions and Pronase digestion do not dissociate the TCE-protein complex . The investigators further concluded from their study that, in order to bind to protein, it was necessary for TCE to be metabolized to its epoxide . The latter reactive intermediate is most likely involved in TCE carcinogensis and toxicity . Fluroxene (Fluomar ) Trifluoroethyl vinyl ether is of particular historical interest in that

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it was the first successfully used fluorinated anesthetic (38) . Blake and coworkers (39) were able to demonstrate metabolism of this anesthetic by using fluroxene with 14C differentially labeled in the vinyl or the trifluoromethyl group . Following intraperitoneal administration of fluroxene to mice and dogs in subanesthetic concentrations, rapid biotransformation occurred (39) . Major metabolites were identified as trifluoroethanol glucuronide, trifluoroacetic acid, and carbon dioxide (from vinyl carbon alone) . Urinary metabolites were collected in the mouse over a 48 hour period, and the measured nonvolatile radioactivity accounted for 9 .3 to 11 .3 of the injected dose, with 14 C02 contributing an additional 2 .1~ metabolism during the first 6 hours . Subsequent studies in intact animals considered the metabolism of fluroxene using anesthetic doses . Studing fluroxene toxicity in mice, Cascorbi and Singh-Amaranath (40) found the followin : (1) mice died within 5 to 24 hours after termination of the anesthetic, (2~ mortality increased directly with anesthetic duration, and (3) phenobarbital pretreatment further increased mortality . The investigations concluded that metabolism of fluroxene to trifluoroethanol was the most likely cause for this toxicity . These workers (40) also observed that post-anesthetic toxicity to fluroxene could be reduced by the prior administration of enzyme inhibitors . Other animal species have been investigated in terms of their response to fluroxene anesthesia . Harrison and Smith (41) examined phenobarbital induced Wistar rats and found massive hepatic necrosis following fluroxene anesthesia . Uninduced animals showed only minor liver changes, and none of these animals died . Johnston and co-workers (42) studied dogs, cats, and rabbits, and a severe toxicity to fluroxene was present in all three species following repeat anesthetic administration . Recent studies by Harrison and co-workers in phenobarbital induced rats have investigated the contribution of various component groups of the fluroxene molecule to toxicity (43) . The trifluoroethyl moiety proved to be the common denominator in toxicity . Neither liver necrosis or mortality followed repeated exposure to vinyl or divinyl ether . Subsequently, Ivanetich and colleagues (44) incubated fluroxene with rat hepatic microsomes and an NADPH generating system . They showed destruction of cytochrome P-450 greater than that observed in the presence of an NADPH gener ating system alone . Apparently the vinyl group of fluroxene is equivalent to an allyl group in its ability to destroy cytochrome P-450 . A number of experimental results indicate that the in vitro destruction of microsomal cytochrome P-450 mediated by fluroxene requires cytochrome P-450 catalyzed metabolism of the anesthetic . Additionally, destruction of cytochrome P-450 by fluroxene is greater in microsomes from animals pretreated with phenobarbital than in controls (45) . Studies by Munson and co-workers of fluroxene metabolism and its associated toxicity in the rhesus monkey are of special interest in that this species may provide a useful model for humans (46) . Like humans, the rhesus monkey showed no evidence of toxicity following three repeat exposures to clinical concentrations of fluroxene for periods of four hours . On the other hand, all animals pretreated with phenobarbital died during their fluroxene exposure . A close association between metabolism and fluroxene toxicity was indicated by an increase of 3 .5 fold in mixed-expired trifluoroethanol concentration, of 2 .5 to 3 .5 fold in trifluoroethanol concentration in blood and urine, and of a 2-fold increase in nonvolatile urinary organic fluoride . In clinical use fluroxene has enjoyed a remarkable history of safety in over 500,00 administrations (47) . However, there are reports of occasional hepatotoxicity in humans . In the two fatal cases of hepatotoxicity reported, both patients had been on enzyme inducing drugs (48,49) . The etiology of hepatic injury in humans following fluroxene has not been defined . Patients with induced hepatic microsomal enzymes may constitute a high risk group for hepatic damage after vinyl radical containing anesthetics (48) .

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Recently the possibility has arisen that fluroxene may be a potential carcinogen . Baden and colleagues (50) have demonstrated by the Ames Salmonella microsomal assay (51) that fluroxene is mutagenic . This is a highly sends fivescreening test for chemicals requiring bioactivation in order to express their mutagenic (carcinogenic?) potential . Over 90~ of known chemical carcinogens have been shown to be mutagenic in this test system . Therefore, the positive mutagenic properties of fluroxene suggest that it poses a significant health hazard as a potential carcinogen (52,53) . Methoxyflura ne

MOF

The inhalation anesthetic, methoxyflurane (MOF), is a particularly instructive example of a volatile anesthetic with toxicity manifest by biotransformation . After introduction in 1960 by Van Posnak and Artusio, this anesthetic was found to possess long-lasting analgesic effects . Due to extreme lipid solubility and structural configuration of methoxyflurane a considerable fraction (70%) undergoes biotransformation in man (54) . In 1966, Crandell and associates (55) reported a high incidence of polyuria following the use of MOF . The question of viscerotoxicity for MOF was not finally answered until a prospective study was completed . The latter investigation indicated MOF was indeed responsible for a diabetes insipidus-like picture (56) . In a subsequent study a group of randomly selected men given MOF were compared with patients receiving halothane anesthesia (57) . This paper unequivocally established that nephrotoxicity after MOF anesthesia existed in man . In 1972, Mazze and Cousins (58) were able to demonstrate a dose-related polyuric nephropathy in Fischer 344 rats . Establishment of this animal model enabled the mechansim of toxicity to be further elucidated . At least 5 different metabolites of MOF have been identified . Of these, inorganic fluoride [F - ] and oxalic acid deserve special attention . Cousins and Mazze (59) reported that the extent of renal dysfunction following MOF anesthesia was directly related to the peak measured serum ionic fluoride concentration . Subclinical toxicity (increased serum uric acid and impaired concentrating ability) was present when serum ionic fluoride exceeded 50 uM . When serum ionic fluoride was 90-120 uM, slight clinical toxicity (serum hypersomolality and hypernatremia, low urinary osmolality, polyuria) was present . Oxalic acid can also be nephrotoxic and may contribute to the overall toxicity of MOF (58) . Finally, since MOF conversion to fluoride is an inducible reaction, it would be expected that levels of this halogen would be increased following anesthesia with MOF in patients pre-treated with inducing drugs . Halothane The metabolism of this potent, non-flammable halogenated hydrocarbon has recently been the subject of a review by Brown and Sipes (60) . The introduction of halothane into clinical anesthesia practice in 1957 was a significant advance . Within three years this agent became the most popular potent inhalation anesthetic enployed in the Western World . By 1963 anecdotal case reports began to appear of unexplained jaundice following halothane anesthesia . These cases manifested certain characteristics : 1)were rare and unpredictable ; 2) seemed to occur more commonly in obese, middle aged subjects ; 3) were indistinguishable pathologically and clinically from viral hepatitis ; and 4) appeared to occur more commonly after a second exposure to the anesthetic . These reported cases prom fed a large nationwide retrospective survey, The National Halothane Study (61~ . That survey concluded that unexplained jaundice following halothane anesthesia was rare (1/30,000) and the overall safety record of the anesthetic was excellent . Primarily, because of an inappropriate animal model of halothane toxicity, hepatologists

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searched for other possible mechanisms of halothane hepatotoxicity . An allergic or hypersensitivity reaction was implicated primarily by exclusion . In 1964, Van Dyke and Chenoweth (62) described halothane biotransformation in vitro . Subsequently this same group reported that halothane metabolism was NCH-02 dependent, occurred primarily in the hepatic microsomal fraction, and was inducible with phenobarbital . Using in vivo methods, Stier (63) measured increased urinary bromide excretion therat in following halothane anesthesia . He also identified trifluoroacetic acid and bromide as urinary end-products of halothane metabolism in rabbits (64) . Subsequent clinical studies by Stier et al . (65), Rehder et al . (66), and Cascorbi and Amaranath (67) demonstrated the presence of nonvolatile halothane metabolites in human urine . On the basis of measurements of urinary fluoride excretion in man, the amount of metabolite eliminated over a 13-21 day period was estimated to be 12 to 24 .8 percent of the halothane absorbed (66,67) . By means of infrared spectra and pa er chromatography methods, Stier (64) suggested that trifluoracetic acid (TFAA~ was the only aliphatic metabolite of halothane in man . Neither of the primary urinary metabolites of halothane (TFAA, Br - ) are implicated in hepatotoxicity . However, the plasma bromide level following halothane anesthesia frequently reached borderline soporific concentrations (> 2mEq/1) (68) . Evidence that the biotransformation of halothane could be a vector in hepatic necrosis has been derived from animal data . Cohen (69) demonstrated that fluorine containing non-volatile metabolites of halothane were bound covalently to liver macromolecules of the mouse after anesthesia in an oxygen envirorment . These persisted for almost two weeks . Although halothane anesthesia in rats pretreated with phenobarbital does not uniformly lead to centrolobular necrosis, covalent binding of 1 wC-halothane metabolites increases over 400 per cent in these induced animals . However, in none of these studies did the covalent binding correlate with necrosis, since no necrosis was produced . Uehleke and co-workers (70) were the first to point out enhanced covalent binding following anaerobic incubation of 14 C halothane with phenobarbital induced rabbit microsomal protein and NADPH . These experiments were felt to give some substantiation to Stier's concept that initiation of halothane biotransformation was dehydrogenation with the formation of the CF3 CC1Br radical or anion . Actually the CF3 CC1Br radical may be less reactive than the debrominated CF3CCIH radical postulated by Van Dyke (71) . Van Dyke's group has investigated several interesting points pivotal to biotransformation of halothane in the presence of reduced 02 concentrations . An initial publication indicated that within the first 2 hrs after administration the covalent binding of 14 C-halothane metabolites to microsomes is greater in the phospholipid component than in protein (71) . It was found that this binding, when carried out in an hypoxic, in vitro environment could be linearly correlated with the formation of diene con~uga et s, precursors of lipid peroxidation . Using both ssCl halothane and 14 halothane, it was determined that the covalent binding of halothane metabolites to microsomal phospholipid retained the chlorine atom . However neither the binding nor the lipid peroxidation was of sufficient intensity to destroy cytochrome P-450 . In summary, these studies, coupled with those of Uehleke's group (70) strongly implied that halothane biotransformation in an anaerobic or hypoxic environment produces reactive intermediates, particularly if metabolism is enhanced by phenobarbital induction . Cohen et al . (72) published a sophisticated investigation of human halothane biotransformation using 14 C-halothane . Urinary nonvolatile metabolites were identified, after separation, by nuclear magnetic reasonance and mass spectroscopy . Three major organic metabolites were found : trifluoroacetic acid, N-trifluoroacetyl-2-aminoethanol, and N-acetyl-S-(2-bromo-2-chloro-1, 1-difluoroethyl)-1-cysteine . The ability of human liver to defluorinate the extremely stable CF3 bond was revealing . The presence of cysteine and ethanolamine conjugates is of concern to human toxicology since these substances imply the presence of reactive intermediates . In all likelihood these conjugated metabolites indicate urinary excretion of degraded hepatic lipid and protein macro-

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molecules coupled covalently with halothane metabolites . Recently, two animal models mimicking the human lesion of "halothane hepatitis" (centrolobular necrosis, elevated SGPT and SGOT, etc) have been produced (73,74) . Microsomal enzyme induction was accomplished with polycholoro biphenyl (PCB) which is known to qualitatively and quantitatively alter cytochrome P-450 . In rats pretreated with PCB's, potent inducers of a wide variety of biotransfornration pathways, a 2 hr exposure to 1 percent halothane in 99 percent oxygen caused classic centrolobular necrosis (73,74) . No evidence of lipid peroxidation was seen with this model . The weakness of this model is that polychlorobiphenyls themselves produce modest liver morphologic changes (sudanophilic vacuole accunulation although no centrolobular necrosis) . It is not clear whether the centrolobular necrosis is due to reactive halothane intermediates or to the s~nnation of two substances with adverse liver affects . Widger et al . (71) found that rats anesthetized with halothane under hypoxic cond~ions (7 percent oxygen) demonstrated the following : 1) an elevated plasma fluoride (enhanced trifluoro bond cleavage) 2) a three-fold increase in the binding of 14C halothane metabolites as compared to halothane anesthesia with 20 percent oxygen ; 3) a microsomal lipid/protein binding ratio of 3 .24 compared to 0 .76 in halothane oxygen anesthetized animals . These investigators postulated that hypoxic atmospheres promote biotransformation of halothane by reductive pathways and that the metabolites produced are potentially more hepatotoxic than those produced via the conventional oxidative pathways . These findings in an hypoxic environment are extremely relevant to clinical cases of "halothane hepatitis" because halothane anesthesia in man significantly decreases hepatic blood flow (75) . From the preceding discussion it is possible to postulate a mechanism for halothane induced liver damage . Key points are that : 1)bioactivation of halothane occurs in the endoplasmic reticulum ; 2) this bioactivation is an inducible pathway ; 3) reactive intermediates are produced which bind to lipids and protein ; and 4) reduced 02 levels promote formation of reactive intermediates . In our laboratory we can now consistently reproduce halothane induced centrolobular liver necrosis in rats . This model uses Phenobarbital pretreatment followed by anesthesia with 1% halothane in 14% 02 for 2 hrs . Within 24 hours, extensive centrolobular necrosis and sharply elevated SGPT levels are observed . Only minimal changes are apparent in animals exposed to hypoxic alone (14% 02) or in non-Phenobarbital pretreated rats anesthetized with halothane in high 02 concentrations . It is known that decreased 02 results in enhanced binding of halothane metabolites to microsomal lipids . Details of the mechanism and identification of non volatile and volatile halothane metabolites produced in this model are currently being pursued . In s~amiary, the useful and safe halogenated inhalation anesthetic halothane apparently possesses a rare, unpredictable complication - hepatic necrosis . Evidence is strong from animal experiments that this could be due to qualitatively and/or quantitatively altered biotransformation to reactive intermediates, particularly in a reductive or oxygen deficient pathway . Covalent binding of halothane metabolites occurs but it is not known if this is of sufficient degree to account for toxic effects . It is possible that these new substances could induce a hypersensitivity phenomenon accelerating hepatic cellular destruction . Enflurane (Ethrane) & Isoflurane ~orane These two methylethyl ethers undergo limited biotransformation . Oxidative metabolism of enflurane and isoflurane is limited by low tissue solubility and chemical structure . For example, isoflurane contains a terminal trifluoromethyl group, plus an ether surrounded by halogens, which makes enzymatic attack less likely . There appears to be no point on the molecule that can be easily attacked and oxidized by the microsomal enzymes . Gradual ether cleavage and ultimate release of the two fluorines on the methyl group account for the

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defluorination (47) . Both enflurane and isoflurane are metabolized to inorganic fluoride (57, 58,76-78) . The question of whether these agents may produce renal toxicity through the fluoride metabolite must be kept in mind (4) . Enzyme induction with phenobarbital in rats enhances defluorination of MOF in vivo and in vitro (80 but does not enhance defluorination of enflurane or iso urane in v o v~ (79; . This paradox may be explained by the large differences in tissu~ubilities among these drugs . Enflurane and isoflurane have oil/gas partition coefficients of 98 and are more rapidly excreted from the body than are anesthetics with higher fat solubilities such as MOF (oil/gas partition coefficient, 970)f~)~ Therefore enflurane and isoflurane interact with the defluorinase enzymes for a shorter time after anesthesia than more lipophilic anesthetics . Substrate availability in this case is the rate limiting step, and induction of defluorinase activity with phenobarbital would not significantly alter the extent of defluorination . By contrast, MOF is likely to be present in sufficient concentrations to permit defluorination at maximun velocity for many hours, even days, into the postanesthetic period . In this case enzyme concentration would be rate limiting and induction would be a significant factor in defluorination (81) . Adler et al . (80) studied in vitro the kinetics of biotransforn~ation of MOF using rathepatic microson>es . 'i~ rate of biotransformation as measured by analysis of metabolites continued to increase even at near saturation concentrations of the anesthetic . No substrate inhibition was observed . Phenobarbital pretreatment enhanced the pathway to inorganic free fluoride metabolite production to a greater extent than the pathway to organic fluorine containing products . It has generally been assured with all drugs that microsomal enzyme induction produces a purely quantitative change in biotransforn~ation . Adler et al . (81) demonstrated that MOF biotransformation not only is quantitatively increased by induction, but also is qualitatively altered by enhancement of the pathway to inorganic fluoride - containing metabolites as determined by direct measurement. Greestein et al . (80) compared the in vitro metabolism of enflurane, isoflurane, and MOF by ~patic microson~es preparec7from the livers of Fischer 344 rats pretreated with phenobarbital and from untreated rats . Pretreatment with phenobarbital increased the defluorinase activities of all three agents in vitro . Comparison of data derived in vitro versus in vivo must be done carefully. Metabolism of the inhalation an~hetics in ~vo is controlled by a rusher of factors, includin the affinity of enzyme ô substrate (Km), the maximal rates of metabolism ~Vmax), and the length of time that the concentration of drug remains at a level sufficient to permit maximun rate of metabolism . The latter depends to an important extent on the lipid solubility characteristics of the anesthetic . In vitro studies are employed to eliminate variables such as hepatic blood flow ~erential, lipid solubilites, biliary excretion, and total body equilibration of anesthetic concentrations of intracellular components . Thus tissue solubility and molecular structure are considered particularly important factors that influence anesthetic metabolism in vivo (80) . In a study of patients withôut renal disease, Cousins and co-workers (82) demonstrated that the metabolism of enflurane to inorganic fluoride was insufficient (mean peak serus level of 22 .2 + 2 .8 uM) to cause clinically significant renal dysfunction . Although the exactpathway for enflurane metabolism has yet to be established, the suspected end products of metabolism are inor anic chloride, inorganic fluoride, and difluoromethoxy-difuoroacetate (82}q . Nevertheless, the question of fluoride induced nephrotoxicity has been raised for enflurane. This query is particularly germane since enflurane has rapidly become a popular anesthetic ; it is today the inhalation agent nast frequently used in many hospitals . Polyuria and deterioration of renal function following enflurane anesthesia have been reported in a patient with a failing transplanted kidney (4) . These investigators postulated that the threshold for fluoride-induced nephrotoxicity may be lower in diseased kidneys, so that comparatively normal peak fluoride concentrations might be toxic .

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Recently, Eichhorn et al . (5) reported renal failure following an uneventful prolonged anesthetic witTi enflurane . The patient had no obvious cause of ischemic renal failure, was not exposed to arty other known neohrotoxin, yet had a remarkably elevated serum fluoride level (greater than 93 uM1 on the second postanesthetic day. This patient had been briefly exposed to enflurane six weeks earlier. Consonant with these findings, German et a1 . (83) investigated enzyme induction by enflurane in man . They measured t~ ûrinary levels of 6-ßhydroxycortisol (6-OHF) and 17-hydroxy corticosteroids (17-OHCS) pre- and post enflurane anesthesia in six healthy adult male volunteers . Increases in 6-OHF relevant to 17-OHCS have been used as a noninvasive measure of microsomal enzyme induction . The ratio of 6-OHF to 17-OHCS increased markedly in 5 of the 6 subjects, indicating that a single prolonged exposure to enflurane can cause induction of the hepatic endoplasmic reticulun. Until further experience provides more data the risk/benefit ratio of enflurane for anesthesia in patients with abnormal preoperative renal function should be carefully considered (5) . Paralleling the above findings are recent data collected in healthy volunteers to determine the nephrotoxicity of inorganic fluoride released by enflurane (84) . Twelve healthy, male, unpremeditated volunteers were exposed to en fl urane-oxygen anesthesia without operation . Urine-concentrating ability was determined by examing their responses to vasopressin before anesthesia and on days 1 and 5 after anesthesia . These subjects were compared to a similar control group anesthetized with halothane oxygen anesthesia . A decrease 1n average maximum urinary osmolality of 264 + 34 mOsm/kg (26 per cent of the preanesthetic value) was present on day 1 aftér enflurane anesthesia, wh :rças sib-, jects anesthetized with halothane had an increase in maximum urinary osmôlality of 120 + 44 mOsm/kg. Enflurane anesthesia resulted in significantly elevated serum fluoride level that r~ernained above 20uM for 18 hours . No elevation in serum fluoride was observed after halothane anesthesia . The authors concluded that the threshold level for inorganic fluoride induced nephotoxicity is lower than previously suspected. Chronic (Trace Anesthetic

Toxici~

The preceding material considered that patients may be at risk from deletrious effects of inhalation anesthetics or metabolites . However, it should be emphasized that epidemiological surveys suggest that operating room personnel may also be at risk . These individuals are chronically exposed to trace concentrations of volatile chemicals and gases, the toxicity of which has yet to be definitely determined . Some anesthetics may be embryotoxic, teratogenic, carcinogenic, and/or mutagenic . This concern was reinforced by a recent release from the National Cancer Institute of the N .I .H . (34) . Trichloroethylene, an inhalation anes thetic, induces hepatocellular carcinoma in mice . An estimated 5,000 medical, dental, and hospital workers are exposed routinely to TCE as an anesthetic gas each year . Additionally, the National Institute for Occupational Safety and Health estimates that annually more than 280,000 industrial workers are exposed to TCE (34) . It has been known since 1945 that TCE is metabolized (7,8) . In fact, the hepatic biotransforniation of TCE to the nonvolatile hypnotic, chloral hydrate engendered speculation that this intermediate was an essential part of the hypnosis produced . Quantitative data obtained in man revealed that the arterial tension was always less than 20% of the inspired tension presumably because of rapid biotransformation (30) . However, all the other general inhalation anesthetics were assumed to be either not metabolized in man or metabolized to a physiologically insignificant degree . Trace levels were felt to be harmless .

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Epidemiolog i c Stu dies Potential hazards of trace anesthetic concentrations in the operating room environment were ignored . By 1967 this apathetic complacency began to be shattered . Hn historical accounting of how evidence developed regardin trace anesthetic toxicity will be presented based on the extensive ASA study ~85) . In initial data accumulated from a Russian study, Vaisman (86) surveyed 303 Russian anesthesiologists and reported an unusually high incidence of headache, fatigue, irritability, and nausea . He also noted that 18 of 31 pregnancies among female anesthesiologists ended in spontaneous abortion . In addition, two of the pregnancies he studied ended in premature delivery and one in congenital malformation . In 1968, Bruce et al . (87) published a study of the causes of death among anesthesiologists ovér ~0 year period . This retrospective survey revealed a trend toward higher than normal incidences of death from reticuloendothelial and lymphoid malignancies . It was based on 441 causes of death among members of the American Society of Anesthesiologists from 1947 to 1966 . The investigators concluded that a prospective study was indicated to obtain more reliable data . A second prospective study by Bruce et al . (88) revealed that death rates, both overall and among junior, active, and ret réd ASA members, were lower than those for the control group . The single exception was the higher than expected rate of suicide among anesthesiologists . In 1969, Li et al .(89) similarly studied the causes of death among chemists ; he found a-Firmer proportion of deaths from cancer than among other professional men . Nearly half the excess cancer deaths were attributable to malignant lymphomas and cancer of the pancreas . The relevance, if any,is that occupationally both chemists and anesthesiologists are chronically exposed to low concentrations of volatile chemicals and gases . From Denmark, Askrog and Harvald (90) reported an increased rate of spontaneous abortion among anesthetists . Approximately 20% of all pregnancies ended in spontaneous abortion, compared with a rate of approximately 10 percent among the same group prior to operating room employment . One year later, Cohen et al . (91) reported a 38 per cent abortion rate among physician anesthetists andâ 30 percent abortion rate among operating room nurses . Abortion rate of ten per cent was noted for control groups of other female physicians or general duty nurses . Miscarriages occurred earlier in gestation in both operating room nurses and anesthetists, compared with control groups . These results suggested a fetal lethality, possibly due to anesthetic gases . Nevertheless, none of the studies incriminated any specific anesthetic agents, nor was a cause-effect relationship established, In 1972, Knill-Jones et al . (92) studied 563 married women doctors . The frequency of spontaneous abôrtion was higher (18 .2 percent) when the anesthetists were working, than when they did not work (13 .7 percent) . The miscarriage rate in the control group was 14 .7 percent . The incidence of congenital abnormalities was also higher when the mother worked, and 12 percent of anesthetists and 6 per cent of the controls suffered involuntary infertility . Subsequen~;ly, Corbett et al . (93,94) surveyed 621 female nurse aa~esthetists in Michigan . The incidéncé of malignancy in the Michigan nurse anesthetists was compared to age adjusted statistics from the Connecticut Tumor Registry . this survey revealed the following : 1) an incidence of malignancy 3X the expected rate, 2) an increased incidence of hirtl~ defects among offspring of the nurse anesthetist, and 3) an increased incidence of congenital abnormalities when th~ixther practiced during pregnancy . All the above studies were of small population groups . The need for a nationwide study of large numbers of operating room personnel became apparent . The American Society of Anesthesiologists with the financial support of the National Institute for Occupational Safety and Health (NIOSH) undertook this study (85) . Fifty thousand operating room professionals were surveyed, with 24,000 unexposed medical and nursing professionals serving as controls . The

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study found 1 .3-2 times the incidence of spontaneous abortion in exposed, female personnel . Women physician-anesthetists suffered the highest risk, followed by nurse-anesthetists . The .incidence of congenital anomalies among live-born offspring of exposed female-physician-anesthetists was double that among offspring of unexposed female physicians . Among exposed nurse anesthetists, the risk of fetal anomalies was 1 .6 times that of their unexposed counterparts . There was also a 25% increase in the risk of congenital abnormalities in children of unexposed wives of male anesthesiologists . Hepatic disease was more fre uent in exposed female respondent groups compared with unexposed controls even after excluding serum hepatitis) . The range of increase was approximately 1 .3 to 2 .2 fold, with p < 0 .04, < 0 .01, and 0 .08 for the three group comparisons . A similar increase in hepatic disease was reported in the exposed male physician anesthetists compared with male pediatricians (p < 0 .01) . Nevertheless, none of the above studies have established a cause-effect relationship . It has been argued that causative factors could include long hours and tension of the operating room, exposure to radiation, and exposure to patients with transmissible viruses . However, based on all the information gathered, the ASA committee concluded that an increase in disease rates in operating room personnel is present . Exposure to waste anesthetic gases in the operating room environment provides a reasonable explanation . Industrial toxicologists have been concerned for many years with the chronic exposure of workers to low concentrations of industrial chemicals and gases, and threshold limit values (TLV) have been established for a number of agents . The first documentation of occupational exposure of operatin room personnel to anesthetic gases was reported in 1969 . Linde and Bruce ~95) described peak levels of 27 ppm halothane and 428 ppm N20 in the operating room .In 1970, Askrog and Peterson (96) reported average concentrations of 85 ppm halothane and 7,000 ppm N20 in the inhalation zone of the anesthesiologist when a non-rebreathing system was used . In 1971, Corbett et al . (97) found levels of methoxyflurane in the operating room ranging frorom X10ppm in the area of the anesthetist, and from 1-2 ppm around the surgeons . Whitcher et al . (98) measured concentrations of halothane in the operating room environment and documented a ten-fold reduction in atmospheric contamination of the operating room through use of appropriate scavenging equipment . Additional studies of atmospheric concentrations of N20 (99) and trichloroethylene (100) in the operating room followed . Another cause for concern involves hospital dental operating rooms . Two studies of ambient gas concentrations during dental surgery indicate that the concentration of halothane in unscavenged rooms may exceed 73 ppm and nitrous Indeed, significant segments of the oxide from 500 to 5,000 ppm (101,102) . dental profession as well as associated nurse anesthetists and dental assistants are occupationally exposed to trace concentrations of anesthetics . In the United States this exposed dental group would exceed 90,000 individuals . Cohen and colleagues (105) reported a retrospective survey of general dental practitioners and oral sur eons with respect to health problems . There was a significant increase (78%~ of spontaneous abortion in the spouses of exposed dentists and a significant increase (156%) in liver disease for exposed dentists . The apparent increased health hazards to the exposed dentist and his offspring strongly suggest that preventative measures should be taken to modify that occupational environment . Experimental St udie s The clinical implications of chronic inhalation of subanesthetic concentrations of anesthetics is not at all clear . Animal studies indicate halothane is capable of inducing its own metabolism (104) . Trace amounts of anesthetic gases and vapors can be found throughout operating suites, (95,104) and anesthetics are found in measureable amounts in exhaled air and blood of anesthesia

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personnel (97,98,105) . Cascorbi et al . (106) established that anesthetists have a faster rate of halothane b~trânsformation than do personnel not working in the operating room enviormient . This observation apparently confirms that halothane can induce its own metabolism in man . Animal experiments reveal that chronic exposure to one anesthetic can induce the metabolism of another anesthetic (107) . Once the possibility of carcinogenicity of anesthetic agents is entertained, it becomes strengthened by comparing the chemical structures of several known carcinogens . The a-chloro ether structures of bis(chloromethyl) ether and chloromethyl ether are thought to be carcinogenic (108) . The anesthetic isoflurane has this same a-chloro ether structure . The ß-fluoro ethers, methoxyflurane and enflurane, must also be suspect since the ß-configuration of bis (chloroethyl) ether has recently been shown to induce hepatomas when fed to rats (109) . It is both interesting and disconcerting to know that vinyl chloride, a known human carcinogen (110,111,112) was once considered for use as an anesthetic . It was discarded because of its myocardial irritant properties . Perhaps even more disquieting is the structural similarity between the anesthetic trichloroethylene (TCE) and vinyl chloride . Mechanism of Carcino9ensis : One of the proposed mechanisms by which vinyl chloride, TCE, and other hepatotoxins and/or hepatocarcino ens induce their lesions is via biotransformation to reactive intermediates free radicals, epoxides, etc) . These inter mediates can then interact with tissue macromolecules (DNA, RNA, protein, phospholipids) . Because of the high reactivity of these intermediates, it is not possible to isolate and quantitate them .directly. An indirect method of assessing their formation is by determining their covalent interaction with the above mentioned tissue macromolecules . For example, Brodie (113) and Gillette (114) were able to relate the biotransformation of br~anobenzene to the reactive intermediate (epoxide) by measuring the formation of a bromobenzene glutathione (GSH) conjugate and the covalent binding of 14 C-bramobenzene to liver protein . When GSH was depleted, the covalent binding to protein correlated with bromobenzene induced centrolobular necrosis of the liver . Similarly, the covalent binding of CC14, CHC13, CBrC13, halothane, acetoaminophen, dimethylnitrosamine (DMN) and many other compounds has been implicated as the mechanism by which they produce liver necrosis or liver carcinogenesis {in the case of DMN) . DMN, a potent hepatocareinogen in many laboratory species, has been shown to alkylate liver DNA (115) . Since many other carcinogens have shown to covalently interact with DNA, it has been proposed that DNA is a molecular receptor for chemical carcinogens (116) . Various DNA adducts have been isolated after the administration of DMN (117), N-acetylamino fluorene (118), 3,4-benzo{a) pyrene (119), and other carcinogens .' All of these adducts have not been identified and it is important to realize that not all covalent interactions with DNA lead to cancer . For example, DMN yields a variety of adducts, but at present only the methylation of the Os of guanine appears to correlate with DMN induced liver cancer (117,120) . However, the covalent interaction of a drug or other xenobiotic with DNA may predict the carcinogenic potential of the compound . This is particularly lmpor~tant when individuals are subject to continuous exposures of trace aoumnts of the chemical as exists in the operating theater, the chemical laboratory, and numerous other occupational settings . Various factors may modify the biotransformation of these chemicals and thus increase the risk of a potential interaction with DNA . For example, use of certain drugs are known to Induce the microsomal drug metabolizing enzymes and thus increase the bioactivation of certain toxins : CC1q (121), bromobenzene {113), CHCIg (29 ) . Similarly Sipes et al . (122) and Maling et al . (123) have shown that a variety of short chain ~ipFiatic alcohols, incl~ing ethanol, greatly enhance the covalent binding of CC14 to liver macromolecules and the

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activity of DMN-N-demethylase, the microsomal en~yrtie responsible for activating DMN to a carcinogen . Thus excess ethanol intake could increase the risk of liver damage and/or carcinogenesis to individuals chronically exposed to these and chemically similar compounds (i .e . anesthetics) . In summary, there is abundant evidence that relates the bioactivation of xenobiotics,including inhalation anesthetics, to highly reactive intermediates . These intermediates covalently interact with tissue macrromolecules and thus alter the integrity of the cells . Such alterations can lead to various tissue lesions : necrosis, hypertrophy, carcinogenesis . Consequently, it is important to know to what extent anesthetic agents undergo bioactivation and the fate of the reactive intermediates that are produced . The widespread use of these drugs in clinical anesthetic practice dictates continued intense studies . Ac knowled~nents Preparation of this review was funded in part by USPHS Grant RO1 CA21820O1 from the National Cancer Institute. The authors are grateful to Ms . Claudia Cramblett for typing the manuscript . REFERENCES 1 . H .F . CASCORBI, ASA Refresher Courses : Toxicity of Anesthetics 3, 63-72 (1975) . 2 . F .M .T . CARNEY, R. VAN DYKE, Anesth . Analg. 51, 135-160 (1972) . 3. R .I . MAZZE, M.J . COUSINS, J .C . KOSEK, Anest~siology 36, 571-587 (1972) . 4 . R .W . LOEHNING, R.I . MAZZE, Anesthesiology 40, 203-2051974) . 5 . J .H . EICHHORN, J . HEDLEY-WHYTE,T .I .STEINMAN, J .M . KAUFMAN, H .L . LAASBERG, Anesthesiology 45, 557-560 (1976) . 6 . B.R . BROWN, JR ., So . Medical Journal 69, 554-556 (1976) . 7. J .F . POWELL, Brit . J . Industr. Med . 2~ 42-145, 1945 8. T .C . BUTLER, J . Pharmacol . Exp . Ther . 97 . 84-9 2 1949 9. A. KAPPAS, A . ALVARES, Scientific Am 236 22-31 (1977) . 10 . W.D . REID, B . CHRISTIE, G . KRISHNA, J~MITCHELL, J . MOSKOWITZ, B .B . BRODIE, Pharmacology 6, 41-55 (1971) . D. JOLLOW, W.Z . POTTER, M. HASHIMOTA, Fed. Proc 31 (abst), 539 (1972) . T.C . BUTLER, J . Pharm. Exp . Ther . 134. 311-31 0 (~61) . T.F . SLATER, Nature 209 36-40 (19~ B.R . BROWN JR ., A.M .~}C~ALYN, Molecular Mechanisms of Anesthesia, edited by B.R . FINK, Progress in Anesthesiology, Yol ._1 ., Raven Press, New York (1975) . 15 . .E .S . REYNOLDS, J . Pharmacol . Exp . Ther . 155, 117 (1967) . 16 . R.O . RECKNAGEL, Pharmacol Rev. 19, 145 (T~7) . 17 . J .A . CASTRO, H. SASAME, H . SUSSF4CN, J .R . GILLETTE, Life Sc 7, 129-139 (1968) . 18 . I .G . SIPES, K. ASGHAR, E . DOCKS, E . BOYKINS, G. KRISHNA, Fea. Proc . 32, 319 (1973) 19 . I .G . SIPES, B .R . BROWN JR ., Anesthesiology 45, 622-628 (1976) . 20 . I .G . SIPES, G . KRISHNA, J .R . GILLETTE, Life~ciences 20, 1541-1548 (1917) . 21 . K.L . SCHOLLER, BR . J . Anaesth 42, 603-605 (1970) . 22 . J . GREEN, The Fat Soluble Vitamins . Edited by H .F . De LUCA, J .S . WUTTIE Madison, Wis ., University of Wisconsin Press (1970) . 23 . N .R . Di LUZIO, Fed. Proc . 32 (8), 1875-1881 (1973) . 24 . E . GRAVELA, B . DIANZANI, Fé6s Letters 9 (2), 93-96 (1970) . 25 . J .R . MITCHELL, D .J . JOLLOW, W.Z . POTTETF, J .R . GILLETTE, B.B . BRODIE, J . Pharmacol . Exp . Ther . 187, 211-217 (1973) . 26 . G .U . CORSINI, I .G . SIPS G . KRISHNA, Fed Proc 31, 548 (1972) 27 . E .GRAVELA, L . GABRIEL, G . UGAZIO . Biochem Pharmacol 20,2065-2070 (1971) . 11 . 12 . 13 . 14 .

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28 . B .J . FRY, T . TAYLOR, D .E . HATHWAY, Arch . Int . Pharmacodyn Ther . 196 . 98101 (1972) . 29 . B .R . BROWN Jr ., I .G . SIPES, A .M . SAGALYN, Anesthesiology 41, 554-561 (1974) 30 . B . SOUCEK, D . VALACHOVA, Brit . J . Industr . Med 17, 60-64 1960) . 31 . J .W . DANIEL, Biochem Pharmacol 12, 795-797 (196 . 32 . K .C . LI-EBMAN, Mol . Pharmacol . 1,239-242 (1965) . 33 . K .C . LIEBMAN, W .J . MCALLISTER,J . Pharmacol . Exp . Ther . ~, 574-578 (1967) 34 . National Cancer Institute, Carcinogenesis Bioassay of Trichloroethylene, U .S . Dept HEW (June 14, 1976) . 35 . A .H . CONNEY, Pharmacol Rev . 19, 317-366 (1969) . 36 . A .H .Y . LU, R . KUNTZMAN, A .H .~ONNEY, Frontiers of Gastrointestinal Research, 2-31, Basel S . Karger, AG (1976) . 37 . B .L . VAN DUREN, S . BANERJEE, Cancer Res 36, 2419-2422 (1976) . 38 . J .C . KRANTZ, C .J . CARR, J . PHARMACOL . Exp. Ther . 108, 488-491 (1953) . 39 . D .A . BLAKE, H .F . CASCORBI, Anesthesiology ~,, 5601970) . 40 . H .F . CASCORBI, A .V . SINGH-AMARANATH, Anesthesiology 37,480-482 (1972) . 41 . G .G . HARRISON, J .S . SMITH, Anesthesiology 39, 619-625 (1973) . 42 . R .R . JOHNSTON, T .H . CROMWELL, E .I . EGER, D . CULLEN, W .C . STEVENS, T . JOAS, Anesthesiology 38, 313-319 (1973) . 43 . G .G . HARRISON, K.M . IVANETICH, L . KAMINSKY, M .J . HALSEY, Anesth . Analg 55, 529-533 (1976) . 44 . K . M . IVANETICH, J .A . MARSH, J .J . BRADSHAW, L .S . KAMINSKY, Biochem Pharmacol 24, 1933-1936 (1975) . 45 . K.M . IVANETICH, J .J . BRADSHAW, J .A . MARSH, Biochem Pharmacol _25, 773-778 (1976) . 46 . E .S . MUNSON, M .H . MALAGODI, R .P . SHIELDS, Clin Pharmacol Ther _18, 687-690, (1975) . 47 . E .N . COHEN, R .A . VAN DYKE, Metabolism of Volatile Anesthetics : Implications for Toxicity, Addison-Wesley Pub . Co ., Reading Mass . (1977) . 48 . E .S . REYNOLDS, B .R . BROWN JR ., VANDAM L .D . New Eng . J . Med . _286, 530-531 (1972) . 49 . W . K . TUCKER, E .S . MUNSON, D .A . HOLADAY, V . FISEROVA-BERGEROVA, B .M . TURNER, Anesthesiology 39, 104-107 (1973) . 50 . J .M . BADEN, M . KELLEY, R .S . WHARTON, B .A . RITT, R .I . MAZZE, Anesthesiology 45~ 695 (1976) . 51 . B.N . AMES . J . MCCANN, E . YAMASAKI, Mutation Res 31, 347-374 (1975) . 52 . J .M . BADEN, M . BRINKENHOFF, R .S . WHARTON, B .A . RITT, V .F . SIMMON, R .I . MAZZE, Anesthesiology 45, 311-318 (1976) . 53 . J .M . BADEN, M . KELLEY, R.S . WHARTON, B .A . HITT, V .F . SIMMON, R .I . MAZZE, Anesthesiology 45, 695 (1976) . 54 . D .A . HOLADAY, S.RUDOFSKY, P .S . TREUHAFT, Anesthesiology 33, 579-593 (1970) 55 . W .B . CRANDELL, S .G . PAPPAS, A . MACDONALD, Anesthesiology 27 591-607 (1966) 56 . R .I . MAZZE, M .J . COUSINS, Can . Anaesth . Soc . J . 20, 64-80973) . 57 . R .I . MAZZE, J .R . TRUDELL, M .J . COUSINS, Anesthesiôlogy 35, 247-252 (1971) . 58 . R .I . MAZZE, M .J . COUSINS, J .C . KOSE K, Anesthesiology _36,571-587 (1972) . 59 . M .J . COUSINS, R .K . MAZZE, JAMA 225, 1611-1616 (1973) . 60 . B .R . BROWN JR ., I .G . SIPES, Bioc em Pharmacol 26, 2091-2094 (1977) . 61 . National Halothane Study, JAMA 197, 775-788 (196) . 62 . R .A . VAN DYKE, A . CHENOWETH, A . VAN POSNAK, Biochem Pharmacol ]3, 12391248 (1964) . 65 (1964) . 63 . A . STIER, Naturwissenschaften 51, , 64 . A . STIER, Biochem Pharmacol 13^1544 (1964) . 65 . A . STIER, H . ALTER, 0 . HESSLER, K . RENDER, Anesth Analg 43, 723-728 (1964) . 66 . K . RENDER, J . FORBES, H . ALTER, 0 . HESSLER, A . STIEN, Anesthesiology _28, 711-715 (1967) . 67 . H . F . CASCORBI, A .V .S . AMARANATH, Anesthesiology 32,480-482 (1970) . 68 . J .H . TINKER, A .J . GANDOLFI, R .A . VAN DYKE, Anesthésiology _37,480-482 (1970) 69 . E .N . COHEN, Anesthesiology 35, 193-303 (1971) .

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70 . H . UEHLEKE, K . H . HELLMER, S . TABARELLI-PIPLAWSKI, Naur~yn-Schmiedelbergs Arch Pharmacol 279, 39-52 (1973) . 71 . L .A . WIDGER, A .~GANDOLFI, R .A . VAN DYKE, Anesthesiology 44, 197-201(1976) 72 . E .N . COHEN, J .R . TRUDELL, H .N . EDMUNDS, E . WATSON, Anesthe~ology 43, 392401 (1975) . 73 . I . G . SIPES, B .R . BROWN JR ., Anesthesiology 45,622-628 (1976) . 74 . E .S . REYNOLDS, MT . MOSLEN, Anesthesiology ~ 19-27 (1977) . 75 . J .L . BENUMOF, JJ . BOOKSTEIN, SAIDMAN L .J ., R . HARRIS, Anesthesiology 4~ 545-551 (1976) . 76 . R .E . CHASE, D .A . HOLADAY, V . FISEROVA-BERGEROVA, L .J . SAIDMAN, F .E . MACK Anesthesiology 35, 262-267 (1971) . 77 . G .A . BARR, M .J . COUSINS, R .I ., MAZZE, B .A . HITT, J .C . KOSEK, J . Pharmacol . Exp . Ther . 188, 257-264 (1974) . 78 . B .A . HITT, .I R . MAZZE, M .J . COUSINS, H .N . EDMUNDS, G .A . BARR, J .R . TRUDELL J . Pharmacol . Exp . Ther . 18 8 257-264 (1974) . 79 . M .J . COUSINS, R .I ., MAZZE,- C . KOSEK, B .A . HITT, F .V . LOVE, J . Pharmacol . Exp . Ther . 190 . 530-54 1 (1974) . 80 . L .R . GREENSTEIN, B .A . RITT, R .I . MAZZE, Anesthesiology 42, 420-424 (1975) . 81 . L . ADLER, B :R . BROWN, JR ., M .F . THOMPSON, Anesthesiology 44, 380-385 (1976) 82 . M .J . COUSINS, L .R . GREENSTEIN, B .A . HITT, R .I . MAZZE, Anesthesiology 44, 496-500 (1976) . 83 . M .L . BERMAN, O .C . GREEN, R .C . CALVERLEY, N .T . SMITH, E .I . EGER, Anesthesiology 44, 496-500 (1976) . 84 . R . I . MAZZE, R . K . CALVERLEY, N .T . SMITH, Anesthesiology 46, 265-271 (1977) . 85 . AMERICAN SOCIETY OF ANESTHESIOLOGISTS, Ad . Hoc . Committee, Anesthesiology 41, 321-340 (1974) . 86 . A.I . VAISMAN, Eksp Khir Anesteziol 3, 44-49 (1967) . 87 . D .L . BRUCE, K .A . EIDE, H .W . LINDE, ~ .E . ECKENHOFF, Anesthesiology _29, 565569 (1968) . 88 . D .L . BRUCE, K .A . EIDE, N .J . SMITH, F . SELTZER, M .H .M . DYKES, Anesthesiology 41, 71-74 (1974) . 89 . F.P . LI, J . F . FRAUMENI, M .A ., MANTEL, US Natl . Cancer Inst . J . _43, 11591164 (1969) . 90 . V ASKROG, B . HARVALD, SAERTYK, Nord . Med . 3, 490-500 (1970) . 91 . E .N . COHEN, J .W . BELVILLE, B . W . BROWN, Anesthesiology 35, 345-347 (1971) . 92 . R .P . KNILL-JONES, D . B . MOIR, L .V . RODRIGUES, Lancet 2 1326 (1972) . 93 . T .H . CORBETT, R . G . CORNELL, K . LIEDING, J .L . ENDRES,~nesthesiology _38, 260-263 (1973) . 94 . T .H . CORBETT, R .G . CORNELL, J .L . ENDRES, L . LIEDING, Anesthesiology 41, 341-344 (1974) . 95 . H . W . LINDE, D .L . BRUCE, Anesthesiology 30, 363-368 (1969) . 96 . V . ASKROG, R . PETERSON, Saertoryk Nord . Med . 83 . 501-504 (1970) . 97 . T .H . CORBETT, G .L . BALL, Anesthesiology 34, 5537 (1971) . 98 . C .E . WHITCHER, E .N . COHEN, J . TRUDELL JR ., Anesthesiology 35,348-353 (1971) . 99 . T .H . CORBETT, Ferti Steril 23, 866-869 (1972) . 100 . T .H . CORBETT, G .C . HAMILTON,M .K ., YOON, J .L . ENDRES, Can Anaesth . Soc . J . 20, 657-678 (1973) . 101 . . L STRUMIN, J .M . STRUMIN, C .C . MALLIDS, Br . Med . J . 4 459 (1973) . 102 . R .I . MILLARD, T .H . CORBETT, J . Oral 5urg 32, 593 (1974 103 . E .N . COHEN, B . W . BROWN, D . BRUCE, H . CASC~BI, T .H . CORBETT, T .W . JONES, C .E . WHITCHER, J . Am . Dental Assoc . 90, 1291-1296 (1975) . 104 . H .W . LINDE, M .L ., BERMAN, Anesth Analg 50, 656-661 (1971) . 105 . D .L . BRUCE, H .W . LINDE, Anesthesiology 3,517-518 (1972) . 106 . B . HALLEN, H . EHRNER-SAMUEL, M . THOMASON, Acta Anaesthesiol Scand 14, 1721 (1970) . 107 . H . F . CASCORBI, D .A . BLAKE, M . HELRICH, Anesthesiology 32, 119-123 (1970) . 108 . R .A . VAN DYKE, J . Pharmacol . Exp . Ther . 154, 364-369 (1966) . 109 . T .H . CORBETT, Environ Res 9 L 211-214 (19~5J. 110 . P .L . VIOLA, A . BIGOTTI, A, CAPUTO, Cancer Res . 31, 516 (1971) .

J

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111 . C . MALTONI, G . LEFEMINE, Environ Res . 7, 387 (1974) . 112 . J .L . CREECH JR ., M .N . JOHNSON, J . Occûp . Med . 16, 150 (1974) . 113 . B .B . BRODIE, W .D . REID, A . K . CHO, I .G . SIPES, ~`. KRISHNA, J .R . GILLETTE, Proc . Nat . Acad . Sci . USA 68, 160-164 (1971) . 114 . J .R . GILLETTE, Biochem Pharmacol 23, 2785-2794 (1974 . 115 . PN .N . MAGEE, E . FARBER, Biochem J .83, 106-114 (1962 . 116 . J .H . WEISBERGER, The Basic Science ~ Poisions, edited by L .J . Casarett and J . Doull, pp . 333-378, MacMillan, N .Y . (1975) . 117 . P .J . O'CONNOR, M .J . CAPPS, A .W . CRAIG, Br .J . Cancer _27, 153-165 (1973) . 118 . E . KREIK, Cancer Res ~ 2042-2045 (1972) . 119 . W .M . BAIRD, R .G . W4RVEY, P . BROOKES, Cancer Res . 35, 54-57 (1975) . 120 . Y .M . GRADDOCK, Biochem et Biophys Acta 321, 202-210 (1973) . 121 . E .S . REYNOLDS, H .J . REE, M .T . MOSLEM, LabInvest 26, 290-299 (1972) . 122 . I .G . SIPES, B . STRIPP, G . KRISHNA, Proc . Soc Expt.Biol . Med . _142, 237242 (1973) . 123 . H .M . MßLING, B . STRIPP, I .G . SIPES, B . HIGHMAN, W . SAUL, M .A . WILLIAMS, Toxicol . and Appl . Pharniacol 33 L 291-308 (1975) .