toxicity of inhalation anaesthetic agents

Br.J. Anaesth. (1978), 50, 665

TOXICITY OF INHALATION ANAESTHETIC AGENTS

E. N. COHEN

Although hundreds of compounds on the chemist'sshelf are known to produce anaesthesia, relatively feware used clinically. The requirements for a clinicallyuseful anaesthetic agent are necessarily stringent andinclude: a wide margin of safety, total reversibility ofanaesthetic effect and an absence of long-term toxicity.In addition, a successful inhalation anaestheticagent must combine the capacity for depression ofthe central nervous system with low levels of inter-ference with respiration and the circulation. Thecentral nervous system effects must be quicklyreversible.

The acute depressant effects produced by theinhalation anaesthetics contrast with the slowerdevelopment of long-term toxicity involving organssuch as the liver, the kidneys or the reproductivesystem. Although such long-term toxicity associatedwith anaesthetics has been recognized for manyyears, in the past it has been considered to occurinfrequently, to be of little importance and of unde-termined aetiology. More recent findings suggestthat not only are these long-term effects serious, butthey occur more frequently than was previouslyrecognized. In addition the aetiological factors arenow beginning to be understood.

RELATIONSHIP BETWEEN ANAESTHETIC

METABOLISM AND TOXICITY

Several reports now suggest there is a significantassociation between the metabolism of anaestheticagents and the development of toxicity. Whilst themajority of drugs are metabolized in the body to lesstoxic derivatives, this is not always so. Occasionally,chemically inert compounds can be transformed intoreactive metabolites which can combine with tissuemacromolecules. These combinations may be toxic.

ELLIS N. COHEN, M.D., Department of Anesthesiology,Stanford University Medical Center, Stanford, California94305, U.S.A.

Portions of this material have been abstracted fromMetabolism of the Volatile Anesthetics: Implications forToxicity (1977) (Eds. E. N. Cohen and R. A. Van Dyke).Menlo Park, California: Addison Wesley PublishingCompany.

Supported by NIH grant OH 00622-01.

0007-0912/78/0050-0665 §01.00

For example, bromobenzene is metabolized to anactive epoxide (Reid et al., 1971; Mitchell, Jollowand Gillette, 1973). A second example is seen withthe hepatic necrosis produced by acetaminophenU.S.P. (paracetamol) (Brodie et al., 1971; Gillette,1974). The centrilobular necrosis produced by thisdrug is enhanced by pretreating animals withphenobarbitone which induces the mixed functionoxidase system: in contrast, liver necrosis is preventedby prior use of an enzyme inhibitor such as piperonylbutoxide (Mitchell, Jollow and Gillette, 1973) orcobaltous chloride (Tephly and Hibbeln, 1971).

A similar relationship between drug metabolismand toxicity would appear to apply to many of ourinhalation anaesthetics. Few anaesthetic agents, withthe exception of chloroform, are capable of producingdirect cellular damage by themselves, but thehepatic and renal toxicity is related to metabolism ina dose-dependent fashion. For example, Scholler(1970) found that the hepatotoxicity of chloroformwas associated with its metabolism. Chloroformanaesthesia in rats pretreated with phenobarbitonewhich induces the liver enzymes led to extensivehepatic necrosis; pretreatment with the enzymeinhibitor disulfiram reduced or abolished the hepaticdamage.

An association between anaesthetic metabolismand toxicity also occurs with other agents. Cascorbiand Singh-Amaranth (1972) observed that, whereas20% of mice anaesthetized with fluroxene for 1 hdied within 5-24 h, if animals were pretreated withphenobarbitone the mortality was 95%. However,pretreatment with carbon tetrachloride which de-pressed the non-specific hepatic enzymes markedlyreduced the mortality following fluroxene anaesthesia.

With halothane, the association between metabo-lism and toxicity has been more difficult to define.The lack of an adequate animal model was a seriousproblem. The important advance was the use ofanimals pretreated with an enzyme-inducing agent.Stenger and Johnson (1972) and Reynolds andMoslen (1974) observed sub-capsular hepatic necrosisfollowing the administration of halothane to ratspretreated with phenobarbitone. More recently,

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666 BRITISH JOURNAL OF ANAESTHESIA

pretreatment of rats with the polychlorinated biphenylAroclor 1254, was found to lead to a widespreadhepatic necrosis following a halothane anaesthetic(Reynolds, Moslen and Szalo, 1976; Sipes and Brown,1976). Within a few hours of anaesthesia the serumtransaminase concentrations were increased andthere were severe morphological changes in thecentrilobular parenchymal cells.

There are convincing reports that the nephro-toxicity seen following methoxyflurane is the result ofthe metabolic production of fluoride ions in bothFischer 344 rats and in man (Mazze, Cousins andKosek, 1972; Cousins, Mazze and Kosek, 1974). Inrats Mazze, Cousins and Kosek (1972) showed thatthe direct injection of sodium fluoride is capable ofproducing renal damage identical to that seen followingmethoxyflurane anaesthesia.

MECHANISMS OF TOXICITY

Three alternative pathways have been proposed tolink metabolism of anaesthetic agents and theirtoxicity: the accumulation to toxic concentrations ofmetabolites normally readily excreted; the productionof reactive intermediates that form irreversible bondswith tissue macromolecules and, finally, the formationof haptens that could produce hypersensitive orimmune responses. In each case metabolism of theagent is implicated.

Excreted metabolitesA number of metabolites of anaesthetic agents

which are excreted in the urine have been identifiedand their potential toxicity determined.

Trifluoroacetic acid is a major metabolite ofhalothane, fluroxene and isoflurane in several animals.However, chronic administration shows it to have alow level of toxicity (Schimmasek et al., 1966;Stier et al., 1972). Its low toxicity following oral,i.v. or i.p. administration is related to its poorabsorption. In the physiological range of pH valuesit is ionized and is thus unlikely to penetrate cellmembranes from the outside.

Trifluoroethanol is present in the urine followingfluroxene anaesthesia and is a suggested (but un-proven) metabolite of halothane. In mice its LD50

varies from 158 to 350 mg kg"1 (Airaksinen andTammisto, 1968; Blake et al., 1969). Trifluoroethanolis produced in only small amounts in humansanaesthetized with fluroxene and has not beendemonstrated after administration of halothane: itseems that it poses little clinical risk (Gion et al.,1974).

Trifluoroacetaldehyde is a suggested urinarymetabolite of halothane which has yet to be isolated inman or experimental animals. When given i.v. or i.p.in mice its LD50 is 600 mg kg"1, which places itbetween trifluoroacetic acid and trifluoroethanol(Airaksinen and Tammisto, 1968).

Inorganic fluoride. This toxic metabolite is releasedin large amounts during and following methoxyfluraneanaesthesia and in lesser amounts with enflurane,halothane and isoflurane. The amount released withhalothane and isoflurane is normally very small.Fluoride concentrations can increase to nephrotoxicvalues (in excess of 40-50 (xmol litre"1) followingmethoxyflurane (Mazze, Shue and Jackson, 1971).The amount of renal damage is related to the fluorideconcentration.

Oxalic acid. There is an increased urinary excretionof oxalic acid following methoxyflurane anaesthesiaand crystalline oxalate deposits have been demon-strated in the kidneys. Although renal toxicity as aresult of tubular obstruction is known to follow highurinary oxalate concentrations, toxicity only occursat concentrations 10 times greater than those foundfollowing the clinical use of methoxyflurane. Therenal lesion produced by methoxyflurane is histo-logically and functionally different from that seenwith oxalate (Cousins, Mazze and Kosek, 1974).

Reactive intermediatesAlthough the halogenated inhalation anaesthetic

agents are normally considered to be unreactive, itappears that under appropriate conditions they canbe metabolized to reactive free radicals capable ofcombining with cellular constituents. In most stablecompounds the electron orbitals of all the atoms arefilled with pairs of electrons of opposite spin. How-ever, some physical or chemical stresses, such asthose induced by metabolism, may disrupt thisbalanced state, producing compounds containing anatom with a single impaired electron in an outerorbit. This type of compound is a reactive free radical.

There are a number of ways in which such a freeradical might be produced by metabolism of ananaesthetic drug. For example, hydroxylation anddehalogenation of halothane by its interaction withthe cytochrome P-450 system could produce areactive acyl halide:

F Br F Br F OII II I II

F-C-C-H!j» F-C-C-OH -HB/F-C-C-C1II II IF Cl F Cl F


TOXICITY OF INHALATION ANAESTHETIC AGENTS 667

It has also been suggested that, under someconditions, cytochrome P-450 is capable of removinga proton to form a reactive carbanion (Ullrich andSchnabel, 1973):

F Br F Br

F - C — C — H ~H+ , F—C— C-I I

F Cl F Cl

By analogy with the work of Van Duuren and hiscolleagues (1972) with alpha-haloethers it is possiblethat the following reaction may occur:

F Br

F—C—C—H -

F Cl

F H

F—C—C-

F Cl

The formation of free radicals and reactiveintermediates is consistent with evidence showingthat the metabolites bind covalently to liver micro-somes and to tissue macromolecules (Uehleke, Hellmerand Tarbelli-Poplawski, 1973; Van Dyke and Wood,1975).

The cellular damage produced as a result of thebinding of a reactive metabolite varies depending onthe molecular aggregate formed. The molecules withthe highest susceptibility for damage include theunsaturated fatty acids and nucleic acids. Fatty acidcomplexes with phosphatidylcholine and phos-phatidylethanolamine are the major components ofthe membranes of liver endoplasmic reticulum andmitochondria. Since inhalation anaesthetic agents arestrongly lipophilic they can be expected to be closely

associated with these lipoprotein membranes. Thelipid constituents are particularly rich in unsaturatedfatty acids and, thus, binding of the reactive inter-mediates would tend to occur within this milieu inthe hepatic endoplasmic reticulum.

The action of the reactive intermediate maycommence with an attack on the alpha-methylenecarbon atom of the unsaturated fatty acid. Thepresence of the double bond weakens the carbon-hydrogen bond adjacent to the unsaturated bond.Free radical intermediates derived from the halo-genated anaesthetics are thus able to initiate peroxida-tive decomposition of the lipids by abstracting allylichydrogen from the alpha-methylene carbon atom,thereby rearranging the position of the double bonds.A subsequent attack by oxygen produces cleavage ofthe radical (fig. 1) (Brown and Sagalyn, 1975).Unless terminated, this oxidative deterioration will betransferred sequentially to adjacent fatty acidcomplexes in a propagated autocatalytic chain(Recknagel and Ghoshal, 1966).

Brown (1972) produced evidence of hepaticmicrosomal lipoperoxidation in the rat following theadministration of chloroform and halothane. Usingrats pretreated with phenobarbitone, he measured theincrease in diene conjugation resulting from lipoper-oxidation in hepatic microsomes. Brown, Sipes andSagalyn (1974) found, in animals pretreated withphenobarbitone, that chloroform anaesthesiaproduced a decrease in the liver content of gluta-thione and an increase in hepatic necrosis. Pretreat-ment with diethyl malate also decreases glutathioneconcentrations and is associated with liver damage. Itappears that, if the tissue concentrations of suchantioxidants reach critically low values, the reactivemetabolites formed from chloroform are no longer

Malonaldchyde andother cleavage products

FIG. 1. Possible mechanism for lipoperoxidation.



quenched and become free to promote tissue damageby lipoperoxidation.

Wood, Gandolfi and Van Dyke (1976) investigatedthe binding of halothane metabolites to rat livermicrosomes incubated in nitrogen. Under suchanaerobic conditions greater diene conjugationoccurred although the formation of malonaldehydeand other "non-peroxide-conjugated-dienes" wasless in the anaerobic than in the aerobic state. Thusit seems that the initial step in lipoperoxidation occursin the absence of oxygen, but peroxidation does notoccur. In aerobic conditions the smaller amount ofbinding of the products of halothane to lipids mayreflect a preferential oxidation of halothane and itsradicals with the ultimate formation of trifluoroaceticacid.

Hypersensitivity and immune responsesIt is possible that the rare hepatic injury seen

following the use of halogenated anaesthetic agentsmight have an allergic or hypersensitivity basis. Thereactions seen are usually considered to represent adelayed or cell-mediated sensitivity produced byspecialized mononuclear cells, thymus-derived lymph-ocytes, or T cells which react with membrane-boundantigens. Whilst it is generally accepted that simplenon-reactive molecules such as the halogenatedanaesthetics cannot be antigens, their metabolites arereactive and capable of forming stable bonds withproteins or other macromolecules.

The presence in vivo of such metabolites which arecovalently bound to macromolecules in the liver hasbeen demonstrated by a number of workers. Rosen-berg and Wahlstrom (1973) attempted to showwhether trifluoroacetic acid and the other "presumed"halothane metabolites such as trifluoroethanol,trifluoroacetaldehyde and trifluoroacetic anhydridecould act as haptens in rabbits immunized with achicken serum globulin complex. Although theirresults suggested that the immune serum formedsimilar precipitins against the complexes, the findingsare not totally convincing. Later studies by Mathieuand his colleagues (1974) using a trifluoroacetylguineapig albumin complex showed that guineapigsdisplay a typical delayed-type hypersensitivity. Thereis therefore evidence to show that anaestheticmetabolites, when complexed to proteins, can act ashaptens. The fact that a delayed skin hypersensitivitycan be induced does not mean that such haptencomplexes are also capable of causing autoimmuneliver destruction.

SPECIFIC ORGAN TOXICITY

A direct relationship between the metabolism ofanaesthetic drugs and their long-term toxicity hasbeen firmly established. A reduction in the metabo-lism of such drugs by pretreatment with enzymeinhibitors or by antioxidants prevents the toxicity ofchloroform and fluroxene in several species. Inaddition, increased toxicity with chloroform, halo-thane, methoxyflurane or fluroxene follows pre-treatment with enzyme induction agents such asphenobarbitone or 3-methylcholanthrene. The aeti-ology of such toxicity may relate to the formation ofeither reactive metabolites which bind to macro-molecules or toxic metabolic end-products such astrifluoroethanol or fluoride ions. Three organ systemsappear to be the major targets: the liver, the kidneysand the reproductive organs.

HepatotoxicityLiver damage produced by chlorinated hydro-

carbons such as carbon tetrachloride and chloroformis well known. Within 3 years of its introduction in1847, Casper reported the first case of delayedchloroform toxicity (cited by Techendorf, 1921).Chloroform can be considered a true hepatotoxin,since its effects on the liver increase with dose andduration of exposure. The necrosis is preceded byfatty changes in the liver cells nearest the centralveins. As the damage increases the necrosis spreadsto involve the whole lobule with the last area ofdestruction being adjacent to the portal tracts.

An alternative explanation to a direct effect wouldbe related to metabolism of the chloroform. Scholler(1970) found that pretreatment with the anti-metabolite disulfiram prevented the hepatotoxicity ofchloroform. Similarly, in the newborn chloroformdoes not damage the liver, presumably as a result ofthe lack of development of the drug-metabolizingenzyme system (Kato and Gillette, 1965). In contrast,phenobarbitone pretreatment significantly increasesthe toxicity of chloroform (Brown and Sagalyn, 1974).The exact mechanism has not yet been defined, butit has been suggested that the damage is produced asa result of the binding of free radicals to cellularconstituents of the liver.

Although one might suspect that most halogenatedanaesthetic agents produce liver damage in anidentical fashion, this does not appear to be the case.Admittedly, each is capable of causing hepatotoxicity,but the incidence and mechanisms are different. Forexample, halothane was introduced only after careful



animal experimentation had indicated it not to bemetabolized and to lack hepatotoxicity. Millions ofanaesthetics later, it would seem that halothane maybe the cause of rare cases of hepatic necrosis. Althoughthis relationship of halothane to liver damage isdifficult to establish in the clinical situation, such arelationship has been found to occur in rats followingpretreatment with the enzyme inducer, Aroclor 1254(Reynolds, Moslen, and Szalo, 1976; Sipes andBrown, 1976). Similarly, there appears to be arelationship between trace-dose concentrations ofhalothane and hepatotoxicity. Stevens and hiscolleagues (1975) exposed rats in a chamber for 4weeks to halothane concentrations equivalent to1/30 MAC. The animals showed hepatotoxicity withvascular degeneration, lipidosis, focal and zonalnecrosis. Similar animals exposed at 1/600 MACshowed no changes, and animals exposed to inter-mediate concentrations exhibited correspondingdegrees of hepatic injury. These findings wereinterpreted as showing that halothane was capable ofproducing injury by a direct hepatotoxic actionthrough the mechanism of anaesthetic metabo-lism.

There is some evidence that personnel working inoperating rooms who are exposed to trace con-centrations of inhalation anaesthetic agents maydevelop hepatotoxicity. Measurable concentrations ofthese gases can be found in all operating rooms wherethey are used (Linde and Bruce, 1969; Whitcher,Cohen and Trudell, 1971). Although the concentra-tions are small and measured in parts per million,continuing exposure can result in the absorption of asignificant amount of anaesthetic. Once these highlylipid-soluble anaesthetics are taken into the bodythey are eliminated relatively slowly. While most areexcreted intact within several hours, significantamounts remain in the body and are slowly bio-degraded over days and weeks. The incidence ofhepatotoxicity in exposed operating room personnelhas been examined in several large surveys; it appearsto correlate with the presence of trace anaestheticgas concentrations and the duration of the operatingroom exposure (Cohen et al., 1974). The increasein hepatic disease among exposed physician anaesthe-tists was found to be approximately twice that of theunexposed control group. A similar study amongstdentists exposed to higher waste anaesthetic gases intheir practice showed an even greater incidence ofhepatotoxicity (Cohen, Brown et al., 1975).

Halothane. Although the precise cause of halothanehepatotoxicity remains to be established, its meta-

bolism appears to play an essential role whether thepresumed cause is hypersensitivity, the accumulationof toxic metabolites, or the conjugation of reactiveintermediates.

The clinical picture of fever, malaise, arthralgiaeosinophilia or lymphocytopaenia supports thediagnosis of a hypersensitivity-type response. Un-fortunately, these signs do not always appear and, inaddition, similar symptoms accompany viral hepatitis.A number of in vitro tests which depend on thedevelopment of cell-mediated sensitivity have beendesigned to diagnose "halothane hepatitis". Suchtests include lymphocyte transformation and theappearance of antimitochondrial antibodies(Rodriques et al., 1969; Paronetto and Popper, 1970).Although both tests were reported to be highlyspecific, subsequent investigators have failed toconfirm these findings.

The presence of a previous exposure to halothanein more than half the reported cases of hepatitis is inaccord with the sensitization hypothesis. However,with the prevalent use of this anaesthetic, multipleexposures would be expected to occur with increasingfrequency. A small number of individuals withprevious episodes of hepatitis have actually undergonea challenge test with halothane (Belfrage, Ahlgrenand Axelson, 1966; Klatskin and Kimberg, 1969).Two well-known cases of anaesthetists challengedwith small doses of halothane attest to the existenceof a hypersensitivity reaction, although the significanceof these reports has recently been questioned(Simpson, Strunin and Walton, 1973). Finally, thereare data which suggest a significant relationshipbetween repeated exposures to halothane and therapidity with which jaundice develops followingsubsequent exposures (Inman and Mushin, 1974).Although there appears to be a decreasing timeinterval for the development of jaundice followingrepeated exposures to halothane, these data havealso come under question. Thus, while it is temptingto postulate a causal relationship between thehypersensitivity response and hepatotoxicity tohalothane, the data are equivocal.

The metabolism of halothane to toxic end-productshas also been suggested as an explanation for the liverdamage. Studies in the mouse and guineapig withparenterally administered trifluoroacetic acid, as wellas with some presumed halothane metabolites,indicate varying degrees of damage (Airaksinen andTammisto, 1968). However, the route of administra-tion employed in these studies may not have allowedthe metabolites to reach certain critical intracellular



areas, which would be directly accessible to metabo-lites produced in vivo. There also appears to be onlylimited toxicity to the final metabolites of halothane.These metabolites, although weakly toxic, mayconceivably accumulate to hazardous concentrationsas a result of repeated administrations of the anaes-thetic, depression of excretory pathways, or increasedproduction of the metabolites following inductionof the drug-metabolizing enzymes.

Of more serious concern are the studies whichindicate covalent binding of metabolites of halothaneto macromolecules within the liver. The reactiveintermediates are able to bind to both lipid andprotein. Gandolfi and Van Dyke (1978) showed thatthe binding to phospholipids is through the fattyacid moiety. This binding is enhanced at loweroxygen tensions, following diethyl maleate-inducedglutathione depletion or after pretreatment of theanimal with a diet rich in polyunsaturated fatty acids.Enzyme induction increased the production ofreactive intermediates and tended to promoteincreased binding. Hypoxia and a reduced anti-oxidant concentration also act to prolong the half-lifeof the intermediates and thus produce increasingbinding.

Recent studies have described the final urinarymetabolites of halothane in man and also indicate thepresumed toxicity of the reactive intermediates(Cohen, Trudell et al., 1975). Although trifluoro-acetic acid has been recognized as the final metabolitewith limited toxicity, it may be that the oxidativeformation of the trifluoroacetyl radical representsthe key step in the metabolism of halothane. There isevidence that this radical may bind covalently withphosphatidylethanolamine, for N-trifluoroacetyl-ethanolamide is present as a urinary metabolite. Themost likely source of the ethanolamide would be thephosphatidylethanolamine, which is a normal lipidconstituent of cell membranes. Presence of the urinarymetabolite suggests conjugation of the radical with thephosphatidylethanolamine in the cell membrane.The urinary metabolite would be produced as aresult of enzymatic cleavage of this molecule. Theaccumulation of this metabolite within the cell couldresult from either the increased production of theradical following increased metabolism of halothaneor the reduction in the amount of the phosphataseenzyme available for cleavage of the conjugatedmolecule.

The appearance of a cysteine conjugate of halothanein the urine is of considerable importance. Theconjugate found in human urine was 2-bromo-2-

chloro-1-l-difluoroethylene. It was suggested thatthis occurred following the reaction of glutathionewith a reductive dehydrofluorination product ofhalothane. The highly reactive bromochlorodi-fluorethylene intermediate could be formed as aresult of proton abstraction from halothane bycytochrome P-450 (Ullrich and Schnabel, 1973).

It has been suggested that gluthathione plays animportant part in the body by its preferential conjuga-tion of the reactive intermediates formed duringmetabolism of foreign compounds. In vivo studies inrats pretreated with phenobarbitone indicate thatanaesthesia with chloroform, but not with halothane,decreases the concentration of glutathione in the liver(Brown, Sipes and Sagalyn, 1974). This latter findingmay represent a species difference. Although theprotective role of glutathione conjugation in manremains to be established, the administration ofnucleophiles such as cysteine, cysteamine anddimercaprol prevents the liver damage produced inmice by paracetamol (acetaminophen) (Boyland andChasseaud, 1967; Judah, McLean and McLean,1970). Theoretically, the use of these agents offers apossibility for the prevention and treatment ofhalothane-induced hepatitis.

The pathways described in the preceding discussionindicate that the metabolism of halothane mayproceed along either an oxidative or a reductive route.Under aerobic conditions, metabolism preferentiallyleads to fluoride, bromide and trifluoroacetic acid.Widger, Gandolfi and Van Dyke (1976) investigatedthe effects of a reduced oxygen tension on themetabolism of halothane. Rats exposed to a 7%inhaled oxygen concentration responded with sig-nificantly increased concentrations of fluoride ionsin the plasma. This increase in dehalogenation underhypoxic conditions was also accompanied by a morethan three-fold increase in the covalent binding ofmetabolites to the microsomal lipids. This greaterbinding during hypoxia supports the hypothesis thatreductive metabolism will produce a more reactivechemical species.

Methoxyfturane. Hepatitis associated with methoxy-flurane has also been reported. Joshi and Conn (1974)reviewed 24 cases. In most respects the clinical aspectsof this syndrome were indistinguishable from thosefound following halothane. Histologically, the lesionis identical with that of viral hepatitis. The majorityof cases were in women. More than half had aprevious exposure to either halothane or methoxy-flurane, and a cross sensitization between these twoanaesthetic agents seems Likely. Although most



authors support an immunological basis for thishepatitis, no patients have been purposely challengedwith methoxyflurane to induce a recurrence. Severalcase reports, however, discuss patients with previoushepatic injury from methoxyflurane who havereceived a subsequent anaesthetic with a recurrence ofthe hepatic injury (Rosander, 1970; Brenner andKaplan, 1971). There is also one report of hepaticinjury following methoxyflurane administered insubanaesthetic concentrations for obstetric analgesiaon each of two occasions (Rubinger, Davidson andMelmed, 1975). At present there are no data whichdefine the specific reactive metabolites of the meth-oxyflurane that might act as possible haptens,although their existence seems possible.

Fluroxene. This anaesthetic drug has enjoyed aremarkable history of clinical safety in over 500 000administrations. However, recent reports suggest theoccasional occurrence of liver damage in man. Twofatal cases have been described in which the patientswere on enzyme-inducing drugs, and in one individualthis included both phenobaritone and diphenyl-hydantoin (Reynolds, Brown and Vandam, 1972;Tucker et al., 1973). The overall toxicity of fluoroxenenoted in several animal species appears to correlatewith its metabolism to trifluoroethanol. However,humans metabolize fluroxene to trifluoroacetic acid,and only a very small amount is present in the urineas trifluoroethanol (Gion et al., 1974). There is noevidence which defines the cause of the hepaticinjury in man following fluroxene anaesthesia;this syndrome does not correspond with the toxicityseen in the experimental animals. However, it doesseem likely that fluroxene toxicity is associated withits metabolism, although perhaps along differentpathways.

NephrotoxicityIn the broadest sense all anaesthetic agents are

nephrotoxic, since they characteristically produce ageneralized depression of renal functions. Thesechanges are largely secondary to effects of anaesthesiaon the cardiovascular, sympathetic and endocrinesystems. Renal blood flow and glomerular filtrationrates usually return to normal within a few hoursafter the termination of the anaesthetic, although theability to excrete a water load may be impairedsomewhat longer. Persistent abnormalities of renalfunction, however, have been associated with theprolonged use of fluorinated anaesthetics.

Methoxyflurane. There is abundant evidence in manto show that renal toxicity is associated with high-

dose methoxyflurane anaesthesia and that thesyndrome of high output renal failure followsmetabolism of the drug to inorganic fluoride ions.Evidence that it is the inorganic fluoride ion whichproduces the nephrotoxicity in man may be seen withthe production of similar abnormalities in the ratfollowing the i.v. injection of sodium fluoride inamounts calculated to produce concentrations similarto those seen with methoxyflurane anaesthesia(Cousins, Mazze and Kosek, 1974).

Although the mechanism by which the fluroide ioncauses renal toxicity is unknown it is likely to berelated to an interference in the transport of sodiumin the proximal convoluted tubules (Kosek, Mazzeand Cousins, 1972). The toxicity may also beproduced from the potent inhibitory effects offluoride ions on enzyme systems involved with cellularenergy transfer (Wiseman, 1970). An additionalpossibility is that inhibition of adenyl cyclase leads tointerference with the action of antidiuretic hormone(Orloff and Handler, 1964). A fourth possibility is thatinorganic fluoride, which is a potent vasodilator,interferes with the counter-current system of thekidney, leading to an increase in medullary bloodflow, washout of sodium from the interstitium andloss of hypertonicity (Caruso, Maynard and DiStefano,1970).

Although ethical considerations preclude studiesin humans, the induction of the drug metabolizingsystems in the rat with phenobarbitone has beenshown to result in an increased production ofinorganic fluoride and an increase in toxicity (Cousins,Mazze and Kosek, 1974). Pretreatment of the ratswith the antimetabolite, SKF 525A-A, was shown todecrease the metabolism of methoxyflurane and todiminish its nephrotoxicity. There can be littlequestion that the renal damage produced by meth-oxyflurane is associated with its metabolism and theproduction of inorganic fluoride ions.

Enflurane, halothane, isoflurane and fluroxene. Sinceeach of these anaesthetic agents liberates fluroide ionsduring its metabolism, concern has arisen as towhether there is an associated nephrotoxicity. Themetabolism of enflurane to inorganic fluoride isconsiderably less than that for methoxyflurane, witha peak serum inorganic fluoride concentrationaveraging 22 jxmol litre"1 following administration ofa clinically anaesthetic concentration (Cousins et al.,1974). This peak concentration is significantly belowthat known to be associated with nephrotoxicity.Nonetheless, at least two case reports have appearedindicating nephrotoxicity following enflurane. In one,



histological evidence of renal damage appeared withonly a modest increase in the serum inorganicfluoride concentration (Loehning and Mazze, 1974).Since this patient had severe pre-existing renaldisease with a failing kidney transplant, the authorssuggested that the toxicity threshold may have beenlowered in the diseased kidney. In the second case itwas reported that, following 6 h of uneventfulenflurane anaesthesia, the serum inorganic fluorideion concentration reached 93 (xmol litre"1 coincidingwith evidence of renal failure (Eichhorn et al., 1976).Since this patient had received an enflurane anaes-thetic 6 weeks earlier, the possibility of enzymeinduction was suggested; no alternative causes forthe renal failure were found.

Isolated cases of renal damage have also beenreported in association with fluroxene anaesthesia(Reynolds, Brown and Vandam, 1972; Tucker et al.,1973). These patients suffered from hepatic involve-ment and in addition were receiving enzyme-inducingdrugs. Under usual clinical conditions, one would notanticipate renal symptoms in conjunction with thelimited release of fluoride seen with this anaestheticagent.

Toxicity to the reproductive organsThe clinical observations of an increase in the

spontaneous miscarriage rate, of a decreased fertility,and an increase in foetal abnormalities amongstchildren of operating room personnel exposed towaste anaesthetic gases suggest an effect of inhalationanaesthetics on the reproductive system (Knill-Jones et al., 1972; Cohen et al., 1974). Epidemio-logical surveys show that exposed female anaes-thetists and their progeny may show a doubling ofspontaneous miscarriage and foetal abnormalityrates when compared with control groups of un-exposed female paediatricians. Whilst there are nodata which confirm a cause-effect relationship, thereremains the possibility that these effects result fromchronic exposure to waste anaesthetic gases. Further-more, it is likely that the metabolism of such anaes-thetics will be causally implicated. Cascorbi, Blakeand Helrich (1970) suggested, in a small-scale study,that anaesthetists metabolized halothane more effi-ciently than did a control group of pharmacists,although the differences were not statisticallysignificant. It is possible that the anaesthetistsunderwent an induction of their microsomal enzymesas a result of the chronic exposure to anaestheticgases in the operating room.

Ghoneim and his colleagues (1975) found evidence

to show that operating room exposure may also exertan inhibitory effect on other enzyme systems. Theirstudy of the kinetics of Warfarin in anaesthesiaresidents showed a prolongation of the primaryhalf-life by 53% following 4 months of operatingroom exposure. Recent studies with radioactive halo-thane indicate an unusually high concentration of thenon-volatile metabolites in the ovaries and testes ofheart transplant donors (Cohen and Van Dyke, 1977).The role of these metabolites in the previouslymentioned toxicity in anaesthetists has not beendefined. Despite the possibly serious implication ofthese studies, much further information is needed toidentify the precise role of anaesthetic agents andtheir metabolites in the toxicity associated with thereproductive system.

ANAESTHETICS AND CARCINOGENICITY

Although there is legitimate concern regarding therelationship between cancer and anaesthesia, theevidence is fragmentary and only limited studies areat present available. Eschenbrenner (1945) reportedthe prodution of hepatomata in mice following therepeated oral administration of chloroform; however,the hepatic lesions appeared only if the anaestheticdosage was large enough to produce liver necrosis.Kunz, Schaude and Thomas (1969) demonstratedthat a different histological picture of rat livercarcinoma resulted from the administratidn of nitro-samines, depending on whether the animals wereanaesthetized with phenobarbitone, halothane ormethoxyflurane. On the other hand, Peraino, Fryand Staffeldt (1971) showed a protective influence ofphenobarbitone on the hepatocarcinogenesis inducedin the rat by 2-acetyl-aminofluorene. Using an in vitromicrobial system, Baden and his group (1976) wereunable to produce mutagenicity in two histidine-dependent strains incubated with halothane inconcentrations ranging from 0.1 to 30%. In anextension of these studies, mutagenicity was alsoabsent following incubation with methoxyflurane,enflurane, trichloroethylene and isoflurane; in con-trast, exposure to fluroxene in concentrations of 0.1and 30% produced a six-fold increase in the numbersof mutant colonies (Baden et al., 1977).

In another approach, Saffiotti (1978) found thattrichloroethylene, when administered in large intra-gastric doses, was capable of producing hepaticcancer in mice in a dose-dependent fashion. Concernover the effects of chronic exposure to inhalationanaesthetic agents led Corbett (1976) to investigatethe offspring of mice exposed transplacentally and



after gestation to sub-anaesthetic concentrations ofisoflurane. The results obtained after 15 monthsindicated a significant increase in hepatic neoplasms,with no increase in the control groups.

Evidence for the direct induction of human cancerby anaesthetic drugs is largely epidemiologic. Atleast two current studies suggest a relationshipbetween female cancer and operating exposure,presumably causally related to the waste anaestheticgases. A limited study by Corbett and his colleagues(1973) showed the incidence of cancer in nurseanaesthetists in Michigan to be three times that ofthe control group. Evidence from a large-scalenational survey (Cohen et al., 1974) showed theincidence of cancer in exposed female physiciananaesthetists to be twice that of unexposed femalepaediatricians. Somewhat smaller increases in thecancer rates were seen in operating room nurses. Ofconsiderable interest is the as yet unexplained findingthat this increase in cancer does not apply to theexposed male. In the exposed women, there was littledifferentiation in the type or location of cancer thatdeveloped, with the exception that the frequencies ofleukaemia and lymphoma were increased three-fold.

The strong alkylating property of certain haloethershas been shown to be closely related to their carcino-genicity (Van Duuren et al., 1969, 1972). Of thesecompounds, bis(chloromethyl)ether was shown to bea strongly active carcinogen. An even higher degree ofcarcinogenicity has been assigned to vinyl chlorideand its reactive by-products (Block, 1974). Theability of both these groups of hydrocarbons to formreactive intermediates is a property shared with manyhalogenated anaesthetic agents.

Although only limited data are available withregard to nitrous oxide there is some evidence that it ismetabolized (Matsubara and Mori, 1968). It isknown to be biologically active and an enzymeinducer (Remer, 1961; Eastwood et al., 1963). Thefollowing equilibrium serves to illustrate the oxidationstates of nitrogen in aqueous solution:

Nitrous oxide is an intermediate ( + ) 1 oxidation stateand could conceivably be metabolized by either anoxidative or a reductive mechanism. There is ampleevidence to indicate the hepatic oxidation of aminesand the reduction of nitro compounds (Mitchard,1971; Weisburger and Weisburger, 1971). If thehuman liver can be shown to be capable of metaboliz-ing nitrous oxide, particularly to nitric oxide or to thenitrite ion, the additional steps converting the latter

compounds to alkylnitrosamines and diazo alkaneshave already been demonstrated (Wolfe and Wasser-man, 1972).

CONCLUSION

It would appear that a number of mechanisms existfor the potential toxicity of volatile anaesthetic agents.Although the pathways are as yet incompletely definedthere can be little doubt that metabolism plays acentral role. It is thus essential that we continue oursearch for new and non-metabolizable anaestheticagents. Investigations into the possible therapeuticuse of antimetabolite and antioxidant drugs wouldseem to be of interest. With persistent efforts we shallmove closer towards the realization of our goal: toprovide the safest possible anaesthetic to each of ourpatients.

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toxicity of inhalation anaesthetic agents

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